JP2002306178A - Decarbamilase complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a 3D-structural coordinates by obtaining a monocrystal of a complex of decarbamilase and a D-N-carbamoyl-α-amino acid, which is a substrate of the decarbamilase, followed by crystal structure analysis with X-ray, and to provide an excellent decarbamilase isomer which is more advantageous for industrial purpose than the natural decarbamilase by designing a molecule with aims of improving the reactivity against a D-N-carbamoyl-α-amino acid, which is a substrate, optimizing the reactive pH, modifying substrate specificity and others by using the 3D-structural coordinates. SOLUTION: The steric structure of the complex of the decarbamilase and a D-N-carbamoyl-α-amino acid, which is a substrate of the decarbamilase, determined by crystal structure analysis with X-ray. The molecular design technique using the steric structure. A method for obtaining a decarbamilase isomer using the technique. Further, a decarbamilase isomer obtained by the technique.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a D-α-amino acid by removing the activity of recognizing and binding to DN-carbamoyl-α-amino acid as a substrate and / or removing the carbamoyl group modifying the amino acid. A mutant of an enzyme having an activity of converting to a carbamoylase (hereinafter, referred to as decarbamylase), and a mutant of decarbamylase and DN-carbamoyl-
It relates to the crystal and three-dimensional structural coordinates of a complex with an α-amino acid. In addition, the present invention relates to the use of the three-dimensional structure coordinates, in particular, the stability of decarbamylase using the three-dimensional structure coordinates, such as heat resistance, organic solvent resistance, resistance to air oxidation, pH activity profile of enzyme reaction, pH The present invention relates to a method for designing an amino acid mutation for altering the activity optimality and improving the catalytic activity. Further, the present invention relates to a method for producing a decarbamylase mutant using the above three-dimensional structural coordinates, the obtained decarbamylase mutant, and use thereof.

[0002]

BACKGROUND OF THE INVENTION Industrial applications of enzymes are limited not only by their high production and purification costs, but also by
In general, due to a series of factors such as denaturation by heat, denaturation by organic solvents, denaturation by air oxidation and their instability due to aggregation due to the hydrophobicity of enzyme molecules,
This is also because the catalytic activity of the enzyme cannot be used effectively. In addition, even if the enzyme has functions and physical properties such as preferable heat resistance, solvent resistance, substrate specificity, cofactor specificity and optimum pH range, its catalytic activity is high enough for industrial use. This is because there are many cases where there is no such information.

[0003] Therefore, it is extremely important to find out the cause of enzyme instability and the mechanism of the manifestation of catalytic activity in order to find a solution for improving an industrial process using an enzyme and making it more useful. Is an important task. on the other hand,
(I) Causes of instability, (II) Measures that can eliminate or reduce instability without changing the catalytic activity of the enzyme, (III) Mechanism of expression of catalytic activity, (IV) Heat resistance of enzyme It is often necessary to accurately present measures that can improve catalytic activity without changing desirable functions and / or physical properties such as solvent resistance, substrate specificity, cofactor specificity and optimal pH range. With difficulty.

[0004] As a method for obtaining an enzyme having improved physical properties or functions for industrial use of an enzyme that catalyzes a useful reaction, a method of chemically or enzymatically treating a gene encoding the enzyme with an artificially random method is used. Add a mutation,
A so-called random screening method for introducing a recombinant DNA containing a mutation into a host cell and screening for an enzyme having a desired function and / or physical property is well known. However, this method has a drawback that the efficiency of obtaining the desired improved enzyme is extremely low because a large number of screenings are performed without knowing the cause of enzyme instability and the mechanism of expression of catalytic activity.

On the other hand, with the progress of structural biology, the tertiary structures of many proteins such as enzymes have been clarified. Functional changes are also being made actively. This method has the advantage that it is much more efficient than the random screening method in the process of obtaining the improved enzyme, but it is necessary to determine a highly accurate protein three-dimensional structure to be subjected to the molecular design method, and Due to the lack of precision, it is often difficult to obtain the desired improved enzyme.

As used herein, the term “protein three-dimensional structure” refers to a three-dimensional structure of a protein having a certain amino acid sequence, which is formed by folding under a certain condition and which is defined by the certain condition and the amino acid sequence. Say. The three-dimensional structure of a protein is determined by, for example, X-ray crystal structure analysis or nuclear magnetic resonance (NMR), and can be described by three-dimensional structural coordinates. “Three-dimensional structural coordinates” refers to proteins,
Coordinates that describe the three-dimensional structure of molecules such as nucleic acids and organic compounds, and can be described in, for example, a Cartesian coordinate system, a dihedral angle coordinate system, a spherical coordinate system, and the like.

[0007] Optically active D-α-amino acids are important compounds as pharmaceutical intermediates, and in particular, D is an intermediate for producing semisynthetic penicillins or semisynthetic cephalosporins.
-Phenylglycine, D-parahydroxyphenylglycine and the like are exemplified as industrially useful compounds.
As a method for producing such D-α-amino acids, a method is known in which the carbamoyl group of the corresponding DN-carbamoyl-α-amino acids is removed to obtain them. Is carried out by a chemical method or a method utilizing an enzymatic reaction of a microorganism. An enzyme called decarbamylase is used to remove the carbamoyl group.

Regarding the modification of the physical properties of decarbamylase, an enzyme-producing strain (E. coli JM109) having improved heat resistance was successfully screened by a random screening method, and a decarbamylase mutant advantageous for industrial use was obtained. (International publication WO94 / 03
No. 613). In addition, a mutant having improved stability by site-directed mutagenesis (Japanese Patent Laid-Open No. 9-17306)
No. 8) has been acquired. However, these examples
Mutants were prepared based only on the primary sequence of amino acids, and did not utilize rational molecular design techniques utilizing the three-dimensional structure of the enzyme.

Regarding the three-dimensional structure of decarbamylase,
Agrobacterium sp. The three-dimensional structure of the native decarbamylase of interest has been determined by the present inventors (T. Nakai et al., (2000), Stru.
cture, 8, 729-737. Japanese Patent Application No. 11-246
797). In addition, regarding the specific three-dimensional structure coordinates of the three-dimensional structure, the protein data bank (PDB, PDB
protein Data Bank, The Rese
arch Collaboratory For St
structural bioinformatics (RC
(SB) is registered) (registration number: 1ER)
Z), this data is incorporated herein. Here, “natural decarbamylase” refers to Agrobac
teriumsp. Enzymes isolated from strain KNK712 (H. Nanba et al., (1998) Biosci.
Biotechnol. Biochem. , 62, 87
5-881).

[0010] Instability factors relating to the physical properties of enzymes, such as denaturation of enzymes by heat, denaturation by organic solvents, denaturation by air oxidation, and aggregation due to the hydrophobicity of enzyme molecules, are as follows:
Many can be deduced from only the three-dimensional structure of the enzyme alone.
On the other hand, in improving the catalytic activity of the enzyme, changing the substrate specificity, changing the cofactor specificity and the pH activity profile, and changing the pH activity optimum, etc., elucidation of factors that solve these from the three-dimensional structure of the enzyme alone, It is difficult to accurately present strategies. One of the measures to solve these problems is to clarify the three-dimensional structure of the enzyme complex between the enzyme and the substrate, inhibitor or cofactor, and analyze the binding mode between the enzyme and the compound that is the substrate. In doing so, it is most important to determine the mechanism of manifestation of catalytic activity.

The three-dimensional structure of the enzyme complex can be determined from the three-dimensional structure of the enzyme alone and the molecular structure of the substrate, inhibitor or cofactor, etc. by using the program DOCK (TJA Ewing et al., (19)
97); Comput. Chem. 18, 1175
-1189) and AUTODOCK (GM Morri)
s et al., (1998). Comput. Chem. 1
9, 1639-1662), it is possible to make predictions using such a docking program. However, even if these techniques are used, the complex can be predicted with sufficient accuracy to apply a rational molecular design method. It is difficult to predict the three-dimensional structure model. In particular, changes in substrate specificity,
In the case of molecular design aiming at improving catalytic activity, the accuracy of the design often depends greatly on the accuracy of the three-dimensional structural coordinates of the enzyme complex.

On the other hand, in order to determine the three-dimensional structure of the enzyme complex, it is necessary to first form an enzyme-substrate complex. For example, in the case of an enzyme that requires a metal ion as a cofactor, special conditions such as using conditions from which the metal ion has been removed, pH, and buffer conditions under which the enzyme activity is significantly reduced without destroying the steric structure of the enzyme are used. Upon finding, it may be possible to form an enzyme-substrate complex. However, since it is generally not easy to form a complex between an enzyme having catalytic activity and a substrate,
In the three-dimensional structure analysis of many enzyme complexes, the three-dimensional structure of the complex between the enzyme and its inhibitor has been determined. In order to understand the catalytic reaction mechanism of an enzyme, it is extremely important to know the three-dimensional structure of the complex with the original substrate, not with the inhibitor.

In the case of decarbamylase, there is no known inhibitor having sufficient and sufficient binding ability for complex formation.
Be an enzyme that does not require metal ions as cofactors,
Until now, complex crystals suitable for X-ray crystal structure analysis have not been obtained due to restrictions such as the pH and buffer conditions under which the enzyme activity is significantly reduced without denaturation.
If these problems can be solved and a single crystal of the decarbamylase-substrate complex can be obtained and the three-dimensional structure of the complex can be determined with high accuracy, precise analysis of the substrate binding mode, and change of the substrate specificity based thereon, It is possible to apply a rational molecular design method aimed at improving the catalytic activity, and it is also possible to quickly and efficiently obtain a modified enzyme that is advantageous for industrial use.

[0014]

SUMMARY OF THE INVENTION In order to solve such problems, the present invention provides a single crystal of a complex of decarbamylase and its substrate, DN-carbamoyl-α-amino acid, and The purpose is to clarify the three-dimensional structural coordinates by line crystal structure analysis. The present invention also provides
Using the three-dimensional structural coordinates of the decarbamylase complex,
An excellent decarbamylase mutation that is more advantageous for industrial use by performing molecular design with the aim of improving the reactivity with respect to the substrate DN-carbamoyl-α-amino acids, optimizing the reaction pH, changing the substrate specificity, etc. It is an object of the present invention to provide a derivative and a method for producing a D-α-amino acid using the obtained decarbamylase mutant.

[0015]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, a mutant in which cysteine at position 171 estimated to be a catalytic residue of decarbamylase was substituted with alanine, That is, a mutant that retains the activity of recognizing and binding to DN-carbamoyl-α-amino acid, but does not have the activity of removing the carbamoyl group modifying the amino acid to convert it to D-α-amino acid. To successfully form a complex with a substrate, DN-carbamoyl-α-amino acid, and obtain a single crystal. The precise three-dimensional structure of the decarbamylase complex was determined, and the present invention was completed.

The present invention relates to a decarbamylase having the amino acid sequence represented by SEQ ID NO: 1 or having the amino acid sequence represented by SEQ ID NO: 2, and having a portion binding to DN-carbamoyl-α-amino acid, or a mutant thereof. For body crystals. In one embodiment, the crystal is obtained crystallographic symmetry has a unit lattice of the rectangular parallelepiped is a space group P2 ₁ 2 ₁ 2 and the unit lattice constants,: a = 67 ±
5 °, b = 136 ± 10 °, c = 68 ± 5 °.

In one aspect, the present invention relates to three-dimensional structural coordinates of a decarbamylase having a moiety that binds DN-carbamoyl-α-amino acid, or a variant thereof. In one embodiment, the three-dimensional structure of the decarbamylase variant having the amino acid sequence shown in SEQ ID NO: 1 can be provided by the three-dimensional structure coordinates shown in Table 1. In another embodiment, the three-dimensional structure of the decarbamylase variant having the amino acid sequence shown in SEQ ID NO: 2 may be provided by the three-dimensional structure coordinates shown in Table 2.

In another aspect, the present invention relates to a crystal of a complex of a decarbamylase having a portion that binds DN-carbamoyl-α-amino acid, or a mutant thereof, and DN-carbamoyl-α-amino acid. About. In one embodiment, the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
DN-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN
Carbamoyl-phenylalanine, DN-carbamoyl-glycine, DN-carbamoyl-histidine,
DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN-carbamoyl-leucine, D
-N-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-proline,
DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine, D
-N-carbamoyl-threonine, DN-carbamoyl-valine, DN-carbamoyl-tryptophan,
And DN-carbamoyl-tyrosine.

In another embodiment, the present invention relates to a decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 and having a moiety for binding DN-carbamoyl-α-amino acid, a variant thereof and DN-carbamoyl-methionine. ,
Crystals of a complex with DN-carbamoyl-valine, DN-carbamoyl-phenylalanine, DN-carbamoyl-alanine, DN-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-phenylglycine were prepared. Can provide. In yet another embodiment, the crystal is obtained crystallographic symmetry has a unit lattice of the rectangular parallelepiped is a space group P2 ₁ 2 ₁ 2 and the unit lattice constants,: a = 67 ± 5Å, b = 136 ± 10 °, c = 68 ±
5 °.

In still another aspect, the present invention provides a decarbamylase having a amino acid sequence represented by SEQ ID NO: 1 and having a moiety that binds DN-carbamoyl-α-amino acid or a mutant thereof, and DN-carbamoyl-α-amino acid. It relates to the three-dimensional structural coordinates of a complex with carbamoyl-α-amino acid.
In one embodiment, the above DN-carbamoyl-
the α-amino acid is DN-carbamoyl-methionine,
The three-dimensional structure of the complex of DN-carbamoyl-valine, DN-carbamoyl-phenylalanine can be provided by the three-dimensional structural coordinates shown in Tables 3-5. In another embodiment, the DN-carbamoyl-α-amino acid is DN-carbamoyl-alanine, DN-carbamoyl-parahydroxyphenylglycine, DN-
The three-dimensional structure of the carbamoyl-phenylglycine conjugate may be provided by the three-dimensional structure coordinates shown in Tables 6-8. Further, in another embodiment, the three-dimensional structural coordinates of the complex and the natural decarbamylase having the amino acid sequence shown in SEQ ID NO: 3 and the three-dimensional structural coordinates of the decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 2 The three-dimensional structural coordinates of the complex constructed by molecular design can also be provided.

In one aspect of the present invention,
A method for designing a decarbamylase variant, wherein the method is any one of claims 7 to 10 and / or 23 to 34.
And designing a decarbamylase mutant having altered physical properties and / or function based on the three-dimensional structural coordinates described in the section. In another aspect, the present invention provides a method for designing a decarbamylase mutant, comprising the steps of: generating a crystal of an enzyme having decarbamylase activity; determining the three-dimensional structure by X-ray crystal structure analysis of the crystal; And a method for designing a decarbamylase mutant having improved physical properties and / or functions based on the three-dimensional structure of the crystal. In yet another aspect, the present invention relates to a method for producing a decarbamylase variant, comprising the steps of: producing a crystal of an enzyme having decarbamylase activity; The process of determining the structure,
A method comprising designing a decarbamylase mutant having improved physical properties and / or function based on the determined three-dimensional structure of the crystal, and producing the decarbamylase mutant. In one embodiment, the three-dimensional structure used in the method of the present invention is a three-dimensional structure of the decarbamylase of the present invention, and a mutant and / or complex thereof. In another embodiment, the step of designing the decarbamylase variant comprises altering the substrate specificity of the enzyme, altering the catalytic activity of the enzyme, improving the stability of the enzyme,
It is aimed at modifying one or more enzyme properties selected from the group consisting of optimizing activity profile, pH activity optimality and altering the water solubility of the enzyme. In yet another embodiment, the method for producing a decarbamylase variant is directed to altering the specific enzyme activity, the pH activity profile, and the pH activity optimization. In another embodiment, the altering the enzymatic properties comprises altering the substrate specificity of the enzyme. In another aspect, the present invention relates to a decarbamylase variant obtained by the method of the present invention.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
As used herein, “decarbamylase activity” refers to D
-N-carbamoyl-α-amino acid as a substrate,
It refers to the activity of binding and / or the activity of removing the carbamoyl group modifying the amino acid to convert it to D-α-amino acid. “Decarbamylase” refers to an enzyme having decarbamylase activity. Examples of decarbamylase include Agrobacterium sp. K
NK712 isolated and amino acid sequenced (supra).

As used herein, amino acids, peptides,
Proteins are represented using abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CBN) shown below. Unless otherwise specified, the sequences of amino acid residues of peptides and proteins are expressed from the left end to the right end so as to be from the N-terminus to the C-terminus, and so that the N-terminus is the first.

A = Ala = alanine, C = Cys = cysteine, D = Asp = aspartic acid, E = Glu = glutamic acid, F = Phe = phenylalanine, G = Gl
y = glycine, H = His = histidine, I = Ile =
Isoleucine, K = Lys = lysine, L = Leu = leucine, M = Met = methionine, N = Asn = asparagine, P = Pro = proline, Q = Gln = glutamine, R = Arg = arginine, S = Ser = serine, T
= Thr = threonine, V = Val = valine, W = Tr
p = tryptophan, Y = Tyr = tyrosine, B = As
x = Asp or Asn, Z = Glx = Glu or G
ln, X = Xaa = any amino acid.

In describing the decarbamylase variants made or contemplated according to the present invention, the nomenclature (original amino acid; position; substituted amino acid) is applied for ease of reference. Therefore, the substitution of cysteine for alanine at position 171 is equivalent to Cys171Al
a or C171A. Multiple mutations are indicated by separating them with a slash symbol ("/"). For example, C171A / V236A is located at position 171
2 shows a mutation that substitutes cysteine for alanine and valine for alanine at position 236.

As used herein, the term “variant” of a mutant of decarbamylase or another enzyme means that at least one amino acid in the amino acid sequence of the original enzyme has been substituted, added, deleted, or modified. A modified enzyme that has an amino acid sequence and retains at least part of the activity of the original enzyme. For example, a variant of decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 is composed of cysteine at position 171 and position 236 of natural type decarbamylase.
Is a double mutant (C171A / V236A) in which valine is substituted with alanine, has an activity of recognizing and binding to DN-carbamoyl-α-amino acid, but modifies the amino acid It has no activity of removing a carbamoyl group to convert to a D-α-amino acid. On the other hand, decarbamylase having the amino acid sequence shown in SEQ ID NO: 2 is a triple mutant of H57Y / P203E / V236A, and has a D-type equivalent to that of natural decarbamylase.
It has an activity of recognizing and binding an N-carbamoyl-α-amino acid and an activity of removing a carbamoyl group modifying the amino acid to convert it to a D-α-amino acid.

The term "active fragment" refers to a fragment having a part of the amino acid sequence of a certain protein enzyme or polypeptide enzyme and retaining at least a part of the activity of the original enzyme. As used herein, “at least a portion of the activity” typically refers to at least 10% of the activity of the original enzyme, preferably at least 50% of the activity of the original enzyme, but if desired, less than 10%. In some cases.

In the present specification, “complex” refers to D-
At an appropriate site in a decarbamylase having a moiety that binds an N-carbamoyl-α-amino acid, or a variant thereof, a stoichiometric equivalent of a substrate, a DN-carbamoyl-α-amino acid or an inhibitor, is added to the non-carbamoylase. A complex molecule that is bound and retained by binding interactions and / or covalent bonds.

As used herein, the term "native crystal" refers to a single crystal in a buffer solution containing only decarbamylase or a variant thereof in a suitable composition containing a precipitant such as ammonium sulfate, polyethylene glycol, and added salts. In one embodiment of the present invention, which refers to crystals that have been grown and do not contain substrates, inhibitors, etc., the crystals of decarbamylase having the amino acid sequence of SEQ ID NO: 1 or 2 are characterized by the concentration and pH of the solution used. Within the specified range (for the concentration of decarbamylase, 1 to 50 mg / ml,
While carefully adjusting the concentration of polyethylene glycol to 5 to 30% by weight and the pH to 6.0 to 9.0), a precipitant solution containing polyethylene glycol (PEG), a buffer, and any added salts was prepared. Can grow. One of three basic techniques commonly used for protein crystal growth, namely, vapor diffusion, dialysis and batch methods (Methods in Enzymology).
y, Volume 114, Diffraction Metho
ds forBiologicalMacromole
cursPartA or Volume 276 Macrom
olecular Crystalgraphy
Although any of Part A) can be used, the vapor diffusion method is preferred.

The vapor diffusion method is a method in which droplets of a protein solution containing a precipitant are placed in a container containing a buffer solution (external solution) containing a higher concentration of the precipitant, sealed, and allowed to stand. is there. Depending on how the drops are placed, hanging-drops
p) Method and sitting drop (sitting)
drop) method. In the hanging drop method, a small droplet of a protein solution is placed on a cover glass, and the cover glass is inverted over a solution reservoir (reservoir) and sealed. On the other hand, in the sitting drop method, an appropriate droplet table is placed inside the reservoir, small droplets of the protein solution are placed on the droplet table, and the reservoir is sealed with a cover glass or the like. The solution in the reservoir contains a precipitant, which is also present in small amounts in the protein droplets. The precipitant solution used in the vapor diffusion method is formed to contain the following components: (a) PEG having a molecular weight of 4000-9000, preferably an average molecular weight of 7500 and a concentration of 10-20% by weight, (b 2.) salt, lithium chloride, magnesium chloride (the best result is obtained with 0.2 M lithium chloride) having a concentration of 0.1-0.5 M as added salt, and (c) pH 6.5-8.0, preferably Is pH 7.5
A sufficient amount of buffer to give 0.05-0.1M H
EPES (Sigma, St Louis, MO, US
A) can be used for this purpose. Other buffers such as sodium phosphate, potassium phosphate and tris (hydroxymethyl) aminomethane malate may also be used.

The "batch method" means that a precipitant solution is added little by little to a protein solution, and when the solution becomes slightly turbid, insoluble substances are removed by centrifugation, the supernatant is placed in a small test tube, sealed, and then left to stand. The way to keep it. The “dialysis method”
The protein solution was added to the buffer solution (external solution) containing the precipitant.
Dialysis using a semipermeable membrane (Methodsi)
n Enzymology, Vol. 114, Diffra
ction Methods for Biological
alMacromoleculesPartA).

The native crystal of decarbamylase having an amino acid sequence represented by SEQ ID NO: 1 or 2 having a predetermined size and suitable for X-ray crystal structure analysis prepared as described above may have a rhombic plate-like outer shape. . Although not suitable for X-ray crystal structure analysis, small or fine crystals having a needle-like or columnar outer shape can be obtained by appropriately selecting conditions such as a precipitant and a buffer solution. In the present specification, the “predetermined size” refers to a minimum size that can be measured by X-ray crystal structure analysis. In the case of the decarbamylase crystal of the present invention, it is preferably 0.3 × 0.3 × 0. .05m
m or more.

In one embodiment of the present invention, there is provided a complex crystal effective for X-ray crystal structure analysis, that is, a crystal in which a substrate, an inhibitor and the like are bound to an enzyme in the crystal. The complex crystal of decarbamylase is prepared by immersing the crystal in a solution containing a substrate, an inhibitor, and the like, which give a required concentration, which allows the native crystal to be stably stored for at least several days without dissolving or disintegrating, and / or Alternatively, a decarbamylase containing a precipitating agent such as ammonium sulfate or polyethylene glycol and an added salt in an appropriate composition, and further, is grown into a single crystal in a buffer containing a substrate, an inhibitor, etc., which give a required concentration. It can be prepared by a crystallization method.

As a decarbamylase used for the preparation of the complex crystal, Cy which is presumed to be one of the catalytic residues is used.
Mutants in which s171 is substituted with Ala, Ser, Thr, Val, Gly or the like, or mutants in which Glu46 and / or Lys126 are substituted with Ala or the like, that is, an activity of recognizing and binding to DN-carbamoyl-α-amino acid , But not having the activity of removing the carbamoyl group modifying the amino acid, for example, a decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 1.

The substrate used for the preparation of the complex crystals of decarbamylase is DN-carbamoyl-α-amino acid, DN-carbamoyl-phenylglycine,
DN-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN
Carbamoyl-phenylalanine, DN-carbamoyl-glycine, DN-carbamoyl-histidine,
DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN-carbamoyl-leucine, D
-N-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-proline,
DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine, D
-N-carbamoyl-threonine, DN-carbamoyl-valine, DN-carbamoyl-tryptophan,
And DN-carbamoyl-tyrosine. In the immersion method, a substrate having a concentration of 0.1 to 50 mM, for example, DN-carbamoyl-methionine, DN
-Carbamoyl-valine, DN-carbamoyl-phenylalanine, DN-carbamoyl-alanine, D-
N-carbamoyl-parahydroxyphenylglycine,
Stock solutions containing suitable precipitants and added salt compositions containing DN-carbamoyl-phenylglycine and the like to provide the desired pH may be used. A preferred example of the storage solution is 15-30% by weight polyethylene glycol 6000.
HEP containing 0.1M and 0.2M lithium chloride
ES buffer (pH 7.5). In the co-crystal method, 0.
Substrates at a concentration of 1 to 50 mM, such as DN-carbamoyl-methionine, DN-carbamoyl-valine, DN
-Carbamoyl-phenylalanine, DN-carbamoyl-alanine, DN-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-phenylglycine and the like, and the concentration and pH of the solution to be used are within specified ranges (decarbamylase). For the concentration of
~ 50mg / ml, polyethylene glycol (PEG)
5 to 30% by weight for pH and 6.
It can be grown from a solution containing polyethylene glycol, a buffer and any added salts, with careful adjustment to 0-9.0).

In general, protein crystals are known to be considerably damaged by X-rays. Therefore, in order to succeed in X-ray crystal structure analysis, it is necessary to obtain crystals that are hardly damaged or to obtain damages. It is important to measure diffraction data under conditions that are difficult to receive. In recent years, it is often performed to obtain high-quality, high-resolution diffraction data by freezing a crystal and measuring diffraction data in a frozen state (Methodsin ENZYMOLOGY Vol. 276, Macromolecular Crystal).
illography, Part A, C.I. W. Cart
er, Jr. And R. M. Sweet, "13" P
radial Cryocrystallogra
phy (DW Rodgers)). Generally, protein crystals are frozen for the purpose of preventing the crystals from being broken by freezing.
A device such as treatment with a solution containing a freeze stabilizer such as glycerol is employed. In the present invention, the native crystals of the decarbamylase mutant and the frozen crystals of the complex are obtained by removing crystallization droplets, crystals in a storage solution or an immersion solution without adding a freeze stabilizer, and directly immersing the crystals in liquid nitrogen. And instantaneously freeze it. The frozen crystal can also be prepared by performing the above operation of instantly freezing the crystal immersed in a storage solution to which a freeze stabilizer has been added.

In order to determine whether the substrate is introduced into the crystal, ie, whether it is bound to the enzyme in the crystal, the diffraction intensity data of the complex crystal prepared by the immersion method and / or the co-crystal method is collected, and the present invention is applied. By the molecular replacement method using the three-dimensional structural coordinates of natural decarbamylase determined by the authors, an electron density diagram is created from the diffraction data of the complex crystal, and the presence or absence of the electron density corresponding to the substrate and inhibitor is determined You can do this by doing

The diffraction data of the mutant crystals of the decarbamylase and the complex crystals of the present invention are, for example, R-AXIS.
It can be measured using IV ++ (Rigaku Denki). Diffraction data measurements on frozen crystals can be performed using a suitable cryosystem to reduce crystal damage and obtain high resolution diffraction data. The measured diffraction image data is R-AXI
Data processing program Crystal Cle attached to S
The reflection intensity data (crystal structure factor) is processed using ar (Rigaku Denki), the program DENZO (Mac Science), or a similar image processing program (or single crystal analysis software). At this stage, a space group and a unit cell constant, which are crystallographic parameters, are determined.

The phase of the crystal structure factor required for the X-ray crystal structure analysis can be estimated by applying a molecular replacement method using the three-dimensional structural coordinates of natural decarbamylase as a reference molecule. Program CNS (AT Brunger, Ya
le University, CCP4 (Briti
sh Biotechnology & Biolog
ical Science Research Cou
(nsil, SERC) to calculate the rotation and translation functions to determine the orientation and position of the molecule. In the analysis of the composite crystal, the electron density is calculated using the obtained phase, and the presence or absence of the electron density corresponding to the substrate DN-carbamoyl-α-amino acid on the electron density diagram is determined at position 17.
Complex formation can be determined by confirming around the active site containing one catalytic residue. The obtained electron density diagram can be used as it is for the construction of a three-dimensional structure model on a three-dimensional graphic. By performing the calculation, an electron density diagram with reduced noise can be obtained.

The three-dimensional structural models of the decarbamylase mutant and the complex were obtained by using program O (program O).
(A. Jones, Uppsala Universi
(Tet, Sweden) from an electron density diagram displayed on three-dimensional graphics by the following procedure.
First, the three-dimensional structure of the reference molecule, natural decarbamylase, is displayed on the electron density map using the obtained rotation and parallel functions. Modify the partial structure of the group. In the case of the complex, the electron density corresponding to the substrate molecule is further found from the electron density diagram, and a three-dimensional structure model of the substrate molecule is constructed so as to match the electron density. By the above procedure, an initial three-dimensional structure model of the whole molecule including the substrate molecule is constructed. The constructed initial three-dimensional structure model is obtained by using the model as a starting model structure and a structure refinement program CNS.
(A.T. Brunger, Yale Univers
According to the refinement protocol of (iii), the three-dimensional structure coordinates describing the three-dimensional structure are refined. This allows
The determination of the stereostructure of the decarbamylase variants and complexes of the present invention is completed.

Structural refinement is calculated from the R factor, which is used by those skilled in the art as an indicator of the accuracy of three-dimensional structural coordinates, and from structural factors that were not independently included in the refinement calculation at the stage of structural refinement. R factor (Free-R
(Factor called factor), and the structure refinement is often completed when the R factor becomes 20% or less.

As for the three-dimensional structure coordinates, the degree to which the R factor is reduced to the point where the refinement of the structure is finished is arbitrary, so that the finally obtained three-dimensional structure coordinates can also be arbitrarily selected. Therefore, in this specification, the structure coordinate homology R is used as an index indicating the difference between the three-dimensional structure coordinates.
Use MSD. “Structural coordinate homology RMSD” refers to the value of the least square deviation (RMSD) representing the similarity of the three-dimensional structure calculated from the three-dimensional structural coordinates of any two molecules. The structural coordinate homology RMSD of a protein is, for example, the program MAPS (G. Lu, J .App
l. Cryst. (2000), 33: 176-18.
It can be obtained by using 3) or the like. When the protein structural topologies are similar between proteins having different amino acid sequences, the structural coordinate homology RMSD often falls within approximately 3.0 °. On the other hand, even in proteins having the same amino acid sequence, the structural coordinates RMSD between the three-dimensional structural coordinates obtained from crystals having different space groups, lattice constants, and the like, are 0. 0 when determined from the atomic group constituting the main chain structure. It can range from 1 to 1.0 °. Further, the present RMSD value may be further increased due to a difference in conformation such as a loop structure on the protein surface.

In the three-dimensional structural coordinates of decarbamylase and its mutants, the atomic group (only Cα atom and / or N, Cα, C, O
Atom), the structural coordinate homology RMSD obtained from 3.
The three-dimensional structural coordinates of decarbamylase and its mutants of 0 ° or less, preferably 1.0 ° or less, more preferably 0.5 ° or less are also within the scope of the present invention. In addition, since the three-dimensional structural coordinates represent the origin of the unit cell in the crystal as the origin in the three-dimensional space, when the three-dimensional structural coordinates of the present invention are used for calculation by a computer, the relative position of each atom is calculated. Without changing the arrangement
New three-dimensional structure coordinates obtained as a result of performing mathematical movement operations such as translation, rotation, and symmetry in a three-dimensional space are also within the scope of the present invention.

Decarbamylase obtained according to the present invention,
Or the three-dimensional structure coordinates describing the three-dimensional structure of the mutant or the complex thereof, according to the notation of the three-dimensional structure coordinates of the protein commonly used by those skilled in the art.
-8. In the table, a data group for four atoms is described in one row, and eight data are assigned to each atom. The first piece of data about atoms is
The atomic order (atomic number), the second is the distinction of atoms (atomic species) in amino acid residues, etc., the third is the distinction of amino acid residues (amino acid name), and the fourth is the distinction of protein chains. The fifth is the position number of the amino acid (corresponding to the residue number in the sequence listing), the sixth, seventh and eighth are the atomic coordinates (X,
Y and Z axes) are shown in Å units.

[0045]

[Table 1] Three-dimensional structural coordinates of the decarbamylase mutant having the amino acid sequence represented by SEQ ID NO: 1 (C171A
/ V236A)

[0046]

[Table 2] The three-dimensional structural coordinates (H57Y /
P203E / V236A)

[0047]

Table 3 Three-dimensional structural coordinates of a complex of a decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 1 and DN-carbamoyl-methionine

[0048]

[Table 4] Three-dimensional structural coordinates of a complex of a decarbamylase mutant having the amino acid sequence represented by SEQ ID NO: 1 and DN-carbamoyl-valine

[0049]

Table 5: Three-dimensional structural coordinates of a complex of a decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 1 and DN-carbamoyl-phenylalanine

[0050]

Table 6: Three-dimensional structural coordinates of a complex of a decarbamylase variant having the amino acid sequence represented by SEQ ID NO: 1 and DN-carbamoyl-alanine

[0051]

Table 7: Three-dimensional structural coordinates of a complex of a decarbamylase variant having the amino acid sequence shown in SEQ ID NO: 1 and DN-carbamoyl-parahydroxyphenylglycine

[0052]

Table 8: Three-dimensional structural coordinates of a complex of a decarbamylase variant having the amino acid sequence shown in SEQ ID NO: 1 and DN-carbamoyl-phenylglycine

The features of the three-dimensional structure of the decarbamylase complex of the present invention are summarized below. (1) The protein structure topology, which is the basic structure, is the same as that of the natural type as shown in FIG. Å0.5 °.

(2) At least Ala71, Glu46, Lys12
6, including the substrate binding mode shown in FIG.

As used herein, the term “protein conformational topology” refers to the geometrical arrangement of secondary structural units of a protein represented by α-helix and β-strand (T.
P. Flores et al., (1994), Prot. En
g. 7, 31-37), “α helix”
Is one of the secondary structures of a protein or polypeptide. One of the most energetically stable structures having a helical structure with a pitch of 5.4 in which amino acids rotate once every 3.6 residues. Say. In the present specification, the “β sheet” is one of the secondary structures of a protein or a polypeptide, and two or more polypeptide chains having a zigzag extended conformation are arranged in parallel, and the peptide Is a structure in which the amide group and the carbonyl group form a hydrogen bond between the carbonyl group and the amide group of the adjacent peptide chain to form an energy-stable sheet. The “parallel β sheet”
Among the β sheets, those in which the amino acid sequences of adjacent polypeptide chains are arranged in the same direction are referred to as “anti-parallel β sheets”. A thing. Further, in the present specification, the “β strand” refers to a single peptide chain having a zigzag-extending conformation forming a β sheet.

The amino acid residues involved in substrate recognition can be identified by examining the interaction such as ionic bond, hydrogen bond, hydrophobic interaction between the substrate molecule and the enzyme molecule from the three-dimensional structural coordinates of the complex. . For example, the α-carboxyl group of the substrate N-carbamoyl D-methionine is represented by A
side chain amide group of sn172, Arg174, Arg17
5 can form an ionic bond or a hydrogen bond with the side chain guanidino group and the side chain hydroxyl group of Thr197. The α-amide group of the substrate is the main chain carbonyl group of Asn196, the carbonyl group of carbamoyl is a side chain amino group of Lys126, the main chain amide group of Asn172, and the amide group of carbamoyl is the main chain carbonyl group of Asn196. It can form a hydrogen bond with a carbonyl group or a side chain carboxyl group of Glu46. Furthermore, the hydrophobic side chains of the substrate N-carbamoyl D-methionine are Pro130, His143, P
ro198, one of the enzyme molecules forming a dimer I
It can interact hydrophobically with nearby residues such as le285.

The three-dimensional structure of the complex of the present invention is obtained by using a decarbamylase mutant having no activity of hydrolyzing an N-carbamoyl group, and is necessary and sufficient for analyzing and understanding the substrate binding mode. Although information can be obtained, Cys171 which is a catalyst residue is replaced with Al.
In order to understand the catalytic reaction mechanism, it is necessary to analyze the interaction between Cys171 and the substrate. Analysis is performed by applying a molecular modeling technique using the three-dimensional structural coordinates of the complex of the present invention and the three-dimensional structural coordinates of the natural decarbamylase and the decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 2. It is possible to easily obtain high-precision three-dimensional structural coordinates of a complex of decarbamylase having a difficult hydrolyzing activity and a substrate.

The three-dimensional structural coordinates of the complex of the present invention are added to the three-dimensional structural coordinates of the natural decarbamylase by the program O
(Supra), Swiss-PDBViewer (Swiss
s Institute of Bioinforma
tics (SIB), ExPASy Molecula
r Biology Server (http: // w
ww. expasy. ch / available from)); Gu
ex, N. And Peitsch, M .; C. (199
7) SWISS-MODEL and theSwi
ss-PdbViewer: An environment
nt for comparative protei
n modeling, Electrophoresis
s 18, 2714-2723) and the like, utilizing the molecular superposition function of a modeling program such as Cys171, which is a catalytic residue, within 12.0 ° of the main chain atom (N, Cα, C And O atom), the least-squares superposition operation can be performed. Using the rotation matrix obtained at that time, the three-dimensional structure coordinates of the substrate molecule in the complex are transformed into the coordinate system of natural decarbamylase, and the transformed three-dimensional structure coordinates are inserted into the three-dimensional structure coordinates of natural decarbamylase. By doing so, a structural model of a complex of decarbamylase having the activity of hydrolyzing a carbamoyl group and a substrate can be constructed. This structural model is stored in the program AMBER (PA Kollman, UCS
F), CHARMM (M. Karplus, Harva
rd University), PRESTO (Morigami et al., Computers Chem. 16, 244-2).
48 (1992)) can optimize the three-dimensional structural coordinates of the complex.
By applying the above operation to the three-dimensional structure coordinates of all six types of substrate complexes whose three-dimensional structure has been determined, the three-dimensional structure coordinates of the complex between the native decarbamylase and the substrate can be obtained. Further, by using the three-dimensional structural coordinates of the decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 2 (Table 2) instead of the three-dimensional structural coordinates of natural decarbamylase, the decarbamylase mutant having catalytic ability and the substrate can be used. And the three-dimensional structural coordinates of the complex with Further, by modeling the side chain of the D-amino acid which is a substrate, the three-dimensional structural coordinates of a complex with a substrate other than the above six types can also be obtained.

By obtaining the three-dimensional structural coordinates of these complexes, it is possible to ascertain the cause of the instability of the enzyme and the mechanism of the expression of the catalytic activity, thereby improving the industrial process using the enzyme and making it more useful. Solutions can be found. Based on the knowledge obtained from the three-dimensional structure of these complexes, a modified design for the purpose of improving the stability of decarbamylase or improving the enzyme activity can be performed. As used herein, “stability” of an enzyme means that the enzyme activity is at least 10% higher than that before heat denaturation even after denaturing the enzyme at a higher temperature (eg, 70 ° C.) than a normal biological environment.
%, Preferably at least 25%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90%.

As used herein, the term “design method” or “molecular design technique” for a mutant molecule refers to analyzing the amino acid sequence and the three-dimensional structure of a protein or polypeptide molecule (for example, a natural molecule) before mutation. Predict the properties (eg, catalytic activity, interaction with other molecules, etc.) of each amino acid, and modify the desired properties (eg, improve catalytic activity, improve protein stability) Etc.) to calculate an appropriate amino acid mutation. This design method is preferably performed using a computer. Examples of computer programs used in such a design method include: programs O (supra), Swiss for programs for three-dimensional graphics, mutagenesis modeling.
s-PDBViewer (supra), InsightII
(Molecular Simulation); Mutation design as a support program, program SHRIKE (Japanese Patent Application No. 11-368498); program for docking of substrate molecules, etc., program DOCK (supra), A
UDOCK (supra); a program A for molecular dynamics calculation and free energy perturbation calculation
MBER (see above), CHARMM (see above), PREST
O (see above).

In the present specification, examples of the amino acid mutation used for designing a mutant include amino acid substitution, amino acid addition, deletion, or modification. Amino acid substitution refers to the replacement of one or more original peptides,
For example, substitution with 1 to 20, preferably 1 to 10, and more preferably 1 to 5 amino acids is meant. The addition of amino acids refers to the addition of one or more, for example, 1 to 20, preferably 1 to 10, and more preferably 1 to 5 amino acids to the original peptide chain. Amino acid deletions refer to one or more, for example, 1
-20, preferably 1-10, more preferably 1
It refers to deleting 5 amino acids. Amino acid modification includes amidation, carboxylation, sulfation, halogenation,
Including, but not limited to, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The substituted or added amino acids may be naturally occurring amino acids, non-naturally occurring amino acids, or amino acid analogs. Natural amino acids are preferred.

The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are in the L-form.

The term "unnatural amino acid" refers to an amino acid not normally found in nature in proteins. Examples of unnatural amino acids include D- or L-forms of norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine, and D-phenylalanine.

"Amino acid analog" refers to a molecule that is not an amino acid, but that is similar in physical properties and / or function to the amino acid. Amino acid analogs include, for example, ethionine, canavanine, 2-methylglutamine and the like. In a further embodiment of the invention, the variant of decarbamylase also comprises an ammonium salt (including an alkyl or aryl ammonium salt), a sulfate, a hydrogen sulfate, a phosphate, a hydrogen phosphate, a dihydrogen phosphate, Peptide salts such as sulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate (methylsulfonate), ethylsulfonate, and benzenesulfonate obtain.

Hereinafter, the mutation of the amino acid of the protein for producing the mutant will be discussed. Methods for performing amino acid substitutions and the like include, but are not limited to, changing the codons of a DNA sequence encoding an amino acid in a technique utilizing chemical synthesis or genetic engineering.

Certain amino acids can be substituted for other amino acids in a protein structure such as, for example, a cationic region or a binding site of a substrate molecule, without any apparent loss or loss of interaction binding capacity. It is the interaction capacity and properties of a protein that define the biological function of a protein. Thus, certain amino acid substitutions may be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Thus, various modifications can be made in the disclosed peptide or the corresponding DNA encoding the peptide without appreciable loss of biological utility.

In designing such modifications, the hydropathic index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyt
e. J and Doolittle, R .; F. J. Mo
l. Biol. 157 (1): 105-132, 19
82). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, which in turn defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5);
Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine ( -1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (- 3.5); lysine (-3.9); and arginine (-4.5)).

It is possible that one amino acid may be substituted by another amino acid having a similar hydrophobicity index, and still yield a protein having a similar biological function (eg, a protein equivalent in enzymatic activity). It is well known in the art. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.
0); aspartic acid (+ 3.0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( -0.4); proline (-0.5)
± 1); alanine (-0.5); histidine (-0.
5); cysteine (-1.0); methionine (-1.
3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-
3.4). It is understood that an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere. In such an amino acid substitution, the hydrophilicity index is preferably within ± 2, and ± 1.
Is more preferably within ± 0.5, and even more preferably ± 0.5.

In the present invention, the term "conservative substitution" refers to a substitution in which the amino acid to be substituted has a similar hydrophilicity index and / or hydrophobicity index as described above. . Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups:
Arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine, and the like.

In molecular design aimed at improving the specific activity of the enzyme and optimizing the optimum pH of the reaction, information on the catalytic reaction mechanism obtained from the three-dimensional structure is extremely useful.
In the present invention, by changing the pKa of the side chain sulfide (SH) group of Cys171 of decarbamylase, it is possible to design an amino acid mutation accompanied by a change in specific activity and pH activity profile and pH activity optimality. Specifically, the calculation of the electrostatic potential of proteins (Takahashi et al., (19
92), Biopolymers 32, 897-90.
According to 9), the effect of the amino acid at each position on the pKa of the SH group can be estimated, and an appropriate amino acid mutation can be designed. For example, an amino acid mutation that makes the electrostatic field near the sulfur atom of Cys171 a more positive electric field, promotes the dissociation of the thiol group (SH) of Cys171, and has little effect on the electrostatic field near the side chain of Glu46 and / or Lys126. Can be designed and selected.

The decarbamylase mutant of the present invention can be designed or prepared by a number of methods other than the methods described above. For example, the sequence of an enzyme having decarbamylase activity may be mutated by oligonucleotide-directed mutagenesis or other conventional (eg, deletion) means at sites identified as desirable for mutation using the present invention. When using the homologous recombination method, DN encoding decarbamylase is synthesized using a synthetic oligonucleotide.
Mutations can be introduced into the A sequence. These oligonucleotides contain the nucleotide sequence adjacent to the desired mutation site. The mutation is the full-length DNA sequence of decarbamylase,
Alternatively, it can be made in any sequence that encodes the fragment polypeptide.

In accordance with the present invention, mutated decarbamylase DNA sequences produced by the above methods or alternative methods known in the art can be expressed using expression vectors.
As is well known in the art, expression vectors typically include an element that enables self-renewal in a host cell independent of the host genome, and one or more phenotypic markers for selection purposes. . Before or after insertion of the DNA sequence surrounding the desired decarbamylase variant coding sequence, the expression vector also contains a promoter, operator, ribosome binding site, translation initiation signal, and, if necessary, a repressor gene or various activator genes and Contains control sequences that encode a termination signal. In some embodiments, if secretion of the produced mutant is desired, a nucleotide encoding the "signal sequence" can be inserted before the decarbamylase mutant coding sequence. For expression under the control of regulatory sequences, the desired DNA sequence must be operably linked to the regulatory sequences. That is, a sequence encoding a decarbamylase variant under the control of a regulatory sequence will have an initiation signal (ie, ATG) to maintain an appropriate reading frame to allow production of an expression product of the sequence. There must be.

A wide variety of well-known available expression vectors are:
All are useful for expressing the mutated decarbamylase coding sequence of the present invention. These include known bacterial plasmids (eg, pBR322), broader host range plasmids (eg, RP4, phage DNA),
chromosomal DNA sequences, such as yeast plasmids, such as μ plasmids or their derivatives, and vectors obtained from combinations of plasmid and phage DNA;
Includes vectors consisting of segments of non-chromosomal DNA sequences and synthetic DNA sequences.

In addition, any of a wide variety of expression control sequences that control expression when operably linked to a DNA sequence may be used in these vectors to express a mutated DNA sequence according to the present invention. You. Such useful expression control sequences include, for example, other sequences known to control viral gene expression and combinations thereof.

A wide variety of hosts are also useful for producing decarbamylase variants according to the present invention. These hosts include, for example, E. coli. Bacteria such as E. coli, Bacillus and Streptomyces, fungi such as yeast, animal cells such as CHO cells, plant cells and transgenic host cells.

It should be understood that not all expression vectors and systems will function in the same manner to express the mutant DNA sequences of the present invention and produce decarbamylase variants. Not all hosts function equally well with the same expression system. However, one of skill in the art can select appropriate vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of the invention.

For example, during selection, the replication capacity of the vector must be considered. Various factors should also be considered when selecting an expression control sequence. They should also take into account, for example, the relative strength of the system, its ability to control, its compatibility with the DNA sequence encoding the decarbamylase variants of the invention, especially with respect to potential secondary structure. The host must be compatible with the selected vector, have the toxicity of the decarbamylase variant to the host, have the ability to secrete the mature product, have the ability to properly fold the protein and form the appropriate conformation, the fermentation requirements, Should be selected based on the ease of purification and safety considerations of the decarbamylase mutants. Within these parameters, one skilled in the art can select various vector / expression control system / host combinations that can produce useful amounts of the mutated decarbamylase.

[0078] The decarbamylase variants produced in these and other systems can be purified by a variety of conventional steps, including those steps used to purify native enzymes having decarbamylase activity. Once a mutation to a decarbamylase is created at a desired location (eg, an active site, a site for stability, or a substrate binding site, etc.), the resulting variant may have any of a number of properties of interest. Can be tested.

For example, variants can be screened for changes in charge at physiological pH. This is determined by measuring the isoelectric point of the decarbamylase variant compared to the isoelectric point (pI) of the decarbamylase before mutation. Isoelectric points are described in Wellner, D .; , Ana
lyt. Chem. 43, 597 (1971).
Is measured by gel electrophoresis according to the method described above. A variant with an altered surface charge is a decarbamylase protein having a pi altered by a substituted amino acid located on the surface of the enzyme, as provided by the structural information of the present invention.

In addition, variants can be screened for higher specific activity compared to the pre-mutation decarbamylase. The activity of the mutant is determined, for example, in Japanese Patent Application No. 11-2467.
It is determined by measuring the ability of DN-carbamoyl-α-amino acids to convert to D-α-amino acids using the assay described in No. 97.

The D-α-amino acids produced by using the decarbamylase mutant of the present invention can be used as pharmaceutical intermediates (for example, D-phenylglycine and D-parahydroxyphenylglycine) as pharmaceuticals (for example, synthetic penicillin). And synthetic cephalosporins).
Pharmaceutical compositions containing the medicaments thus produced can also be provided according to the present invention. Pharmaceutical compositions may contain any pharmaceutically acceptable auxiliary ingredients including excipients, stabilizers, carriers and the like.

The D-α-amino acids produced by using the decarbamylase mutant of the present invention can be used as an agricultural chemical intermediate (for example, D-valine) for producing an agricultural chemical (for example, fluvalinate). A pesticidal composition containing the pesticide thus produced can also be provided according to the present invention.
As used herein, the agricultural composition comprises excipients, stabilizers,
It may contain any agriculturally acceptable auxiliary component such as a carrier.

The D-α-amino acids produced using the decarbamylase variant of the present invention are used as food additive intermediates (for example, D-alanine and D-aspartic acid) as food additives (for example, Alitame). ). According to the present invention, a mutant of a polypeptide enzyme or a protein enzyme similar to the amino acid sequence of decarbamylase obtained by using the above method can be provided.
Mutants of these enzymes can be used in place of the wild-type enzyme, for example, in bioreactors and the like.

According to the present invention, there can also be provided an electromagnetic storage medium in which a program of a three-dimensional structural coordinate of the above molecule such as decarbamylase and / or a molecular designing technique and / or a modification method is recorded. Examples of the electromagnetic medium include a magnetic tape, a magnetic disk, a magneto-optical disk, and an optical disk medium. The present invention will be described in more detail with reference to the following Examples and Test Examples, which are provided for illustrative purposes of the present invention and do not limit the present invention in any way.

[0085]

EXAMPLES The reagents used in the following examples were Nacalai Tesque, Wako Pure Chemical, or SIG, except where noted.
Obtained from MA (St Louis, MO, USA). [Test Example 1] Characteristics of decarbamylase mutant The hydrolysis activity of the decarbamylase mutant having the sequence described in SEQ ID NO: 1 was determined by Nanba H. et al., Biosci. Bi.
Measured according to otechnol. Biochem. 62 (5), p.875-881 (19998).

N-carbamoyl-D-α-parahydroxylphenylglycine was added in an amount of 1.0% by weight.
1 ml of substrate solution dissolved in 1M phosphate buffer pH 7.0
Then, 0.1 ml of a carbamylase mutant enzyme solution of about 0.3-0.4 mg / ml was added thereto, reacted at 40 ° C. for 20 minutes, and 0.25 ml of 5N sulfuric acid was added to stop the enzyme reaction. Was calculated by quantifying the converted D-α-parahydroxyphenylglycine by high performance liquid chromatography using D-phenylalanine as an internal standard solution. As a result, D-α parahydroxyphenylglycine was not detected.

Example 1 Preparation of Decarbamylase Mutant Crystal (I) Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 1 and obtained at a concentration of 10 to 30 mg / ml (0.1 M HEPES buffer, pH 7.0).
5) 5 μl and 15-20% by weight polyethylene glycol 6000 (manufactured by Nacalai Tesque) as a precipitant,
5 μl of a 0.1 M HEPES buffer solution containing 0.2 M lithium chloride, pH 7.5 (manufactured by SIGMA) was mixed on a dropping table, and 500 to 1000 μ of a solution having the above precipitant composition was mixed.
After sealing 1 as a reservoir solution, crystallization was performed at 20 ± 5 ° C. by a vapor diffusion method. After the start of crystallization, 0.3 × 0.3 × 0.05 mm to 1.2 ×
Crystals grew to dimensions of 0.6 × 0.2 mm.

Example 2 Preparation of Decarbamylase Mutant Crystal (II) Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
1 (see 1998)), a decarbamylase solution (0.1 M HEPES buffer, pH 7.5) prepared by preparing decarbamylase having the amino acid sequence represented by SEQ ID NO: 2 and having a concentration of 10 to 30 mg / ml.
μl and 15-20% by weight polyethylene glycol 6000 (manufactured by Nacalai Tesque) as a precipitant, 0.2M
0.1 M HEPES buffer containing lithium chloride, pH
Mix 5 μl of 7.5 (manufactured by SIGMA) on the drop table,
After sealing as a 500-1000 μl reservoir solution having the above precipitant composition, 20 ± 5 by a vapor diffusion method.
Crystallization was performed at ° C. After the start of crystallization, 0.3 × 0.3 × 0.05 mm to 1.2 × 0.6 ×
The crystals grew to a size of 0.2 mm.

Example 3 Collection of X-Ray Diffraction Data of Decarbamylase Mutant Crystal (I) The decarbamylase mutant crystal prepared in Example 1 was charged into liquid nitrogen and frozen, and then a cryosystem (manufactured by Rigaku Corporation) Liquid nitrogen gas sample spraying device), cooled to -170 ° C, R-AXIS IV ++ (manufactured by Rigaku Corporation), X-ray power 45 kV, Cu-Kα generated at 100 mA
Diffraction data measurements were performed using the lines. The diffraction intensity was calculated by using an imaging plate (IP) set at a crystal-detector distance of 120 mm, oscillating at 1.5 ° per frame, and diffracting data for 60 frames with 3 minutes exposure at 1.8 ° resolution. Collected. The measured diffraction image data is stored in a data processing program (Cr
The crystallographic parameters were determined and converted to reflection intensity data (crystal structure factor) using Ystal Clear. The crystallographic parameters are as follows: space group P2 ₁ 2 ₁ 2, unit cell constant a = 67.3 °, b = 136.8 °, c = 6
It was 7.8Å. For the terms such as the space group and the unit cell constant, refer to the introduction to X-ray analysis (Masao Kadoto et al., Tokyo Chemical Dojin).

Example 4 Collection of X-ray Diffraction Data of Decarbamylase Mutant Crystal (II) The decarbamylase mutant crystal prepared in Example 2 was put into liquid nitrogen and frozen, and then the conditions described in Example 3 were used. In this procedure, crystallographic parameters and reflection intensity data were obtained.
The crystallographic parameters are the space group P2 ₁ 2 ₁ 2 and the unit cell constants are a = 67.5 °, b = 137.0 °, and c = 68.2 °.
Met.

Example 5 Determination of Stereostructure of Decarbamylase Mutant Crystal (I) Crystallographic parameters and reflection intensity data of the decarbamylase mutant crystal having the amino acid sequence shown in SEQ ID NO: 1 obtained in Example 3 In contrast, a molecular replacement method using the crystal structure of natural decarbamylase (registration number: 1ERZ) as a reference molecule was applied. The molecular replacement method uses the program CN
The rotation function and the translation function were calculated using S, and the orientation and position of the molecule were determined. Using the obtained phase, the electron density of the decarbamylase mutant was determined. Next, program O (A. Jones, Uppsala Univer)
sitet, Sweden) to display the three-dimensional structural coordinates of the reference molecule, natural decarbamylase, on the electron density diagram displayed on the three-dimensional graphics, correct the model of the site where the amino acid was substituted, and do not fit the electron density Amino acid residue partial structure was modified. Using the initial three-dimensional structure model thus constructed as a starting structure, a structure refinement program CNS (AT Brunger,
The three-dimensional structure coordinates were refined according to the refinement protocol of Yale University. In the refinement, the operation of correcting the local structure having a large deviation from the electron density map, identifying the electron density corresponding to the water molecule, and including the water molecule in the refinement calculation is repeated, and the R value and the free R value (free
Using the R-factor as an index, the refinement was advanced until the R value became 20% or less. R of the final model structure with water molecules added
The value was 18.9% with respect to the reflection data at 69.1-1.8 ° resolution.

Example 6 Determination of Stereostructure of Decarbamylase Variant Crystal (II)
Was determined according to the procedure described in 1. The R value of the final model structure to which water molecules were added was 18.9% with respect to the reflection data with a resolution of 69.1-1.8 °.

Example 7 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-Methionine Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 and obtained in a concentration of 15 to 30 mg / ml (0.1 M HEPES buffer, pH 7.5). A substrate, DN-carbamoyl-methionine, was added, 5 μl of a solution prepared to a concentration of 5 mM, and 15 to 20% by weight polyethylene glycol 6000 as a precipitant.
(Nacalai Tesque), 5 μl of 0.1 M HEPES buffer containing 0.2 M lithium chloride, pH 7.5 (Sigma) were mixed on a dropping table, and 500 to 1000 μl of the solution having the precipitant composition was mixed. After sealing as a reservoir solution, crystallization was performed at 20 ± 5 ° C. by a vapor diffusion method. 0.3 × 0.3 in about 2 days to 2 weeks after the start of crystallization
The crystal grew to a size of × 0.05 mm to 1.2 × 0.6 × 0.05 mm.

Example 8 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-Valine Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 obtained by the method described in No. 1 (1998)) into a decarbamylase solution (0.1 M HEPES buffer, pH 7.5) prepared at a concentration of 15 to 30 mg / ml. A substrate, DN-carbamoyl-valine, was added, and 5
5 μl of solution prepared to mM concentration and 15
0.1M containing -20% by weight polyethylene glycol 6000 (manufactured by Nacalai Tesque) and 0.2M lithium chloride
5 μl of HEPES buffer solution, pH 7.5 (manufactured by Sigma) was mixed on a dropping table, and the solution having the precipitant composition 500 to 500 μl was mixed.
After sealing 1000 μl as a reservoir solution, crystallization was performed at 20 ± 5 ° C. by a vapor diffusion method. 0.3 × 0.3 × 0.05 in about 2 days to 2 weeks after the start of crystallization
The crystal grew to a size of mm to 1.9 × 0.8 × 0.05 mm.

Example 9 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-phenylalanine Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 and obtained in a concentration of 15 to 30 mg / ml (0.1 M HEPES buffer, pH 7.5). A substrate, DN-carbamoyl-phenylalanine, was added, 5 μl of a solution prepared to a concentration of 5 mM, and 15 to 20% by weight of polyethylene glycol 600 as a precipitant.
0 (manufactured by Nacalai Tesque), 5 μl of 0.1 M HEPES buffer containing 0.2 M lithium chloride, pH 7.5 (manufactured by Sigma) are mixed on a dropping table, and 500 to 1000 μl of a solution having the above precipitant composition is mixed. Was sealed as a reservoir solution, and then crystallized at 20 ± 5 ° C. by a vapor diffusion method. 0.3x in about 2 days to 2 weeks after the start of crystallization
0.3 × 0.05mm to 1.8 × 0.8 × 0.05mm
The crystal grew to the size of.

Example 10 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-alanine 20 crystals of the decarbamylase variant prepared in Example 1 were prepared.
% Polyethylene glycol 6000 (manufactured by Nacalai Tesque), 0.1 M HEPES buffer containing 0.2 M lithium chloride, 5 mM DN-carbamoyl-alanine,
A composite crystal was prepared by immersing in 1 ml of pH 7.5 (manufactured by SIGMA) at 20 ± 5 ° C. for 1 to 7 days.

Example 11 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-parahydroxyphenylglycine (I) Twenty-five decarbamylase mutant crystals prepared in Example 1 were used.
% Polyethylene glycol 6000 (manufactured by Nacalai Tesque), 0.1 M containing 0.2 M lithium chloride, 5 mM DN-carbamoyl-parahydroxyphenylglycine.
M HEPES buffer, pH 7.5 (manufactured by SIGMA)
A composite crystal was prepared by immersion in 1 ml at 20 ± 5 ° C. for 1 to 7 days.

Example 12 Preparation of Complex Crystal of Decarbamylase Mutant and DN-carbamoyl-phenylglycine (I) Twenty-five decarbamylase mutant crystals prepared in Example 1 were used.
% Polyethylene glycol 6000 (manufactured by Nacalai Tesque, Inc.), 0.1 M HEPES containing 0.2 M lithium chloride, 5 mM DN-carbamoyl-phenylglycine
Buffer solution, pH 7.5 (manufactured by SIGMA) 1 ml, 20
A composite crystal was prepared by immersion at ± 5 ° C. for 1 to 7 days.

Example 13 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-parahydroxyphenylglycine (II) Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 obtained by the method described in No. 1 (1998)) into a decarbamylase solution (0.1 M HEPES buffer, pH 7.5) prepared at a concentration of 15 to 30 mg / ml. A solution prepared by adding DN-carbamoyl-parahydroxyphenylglycine as a substrate to a concentration of 5 mM and adding 5 μl of the solution was prepared.
0.1M HEPES buffer containing 15-20% by weight of polyethylene glycol 6000 (manufactured by Nacalai Tesque) as a precipitant, 0.2 M lithium chloride, pH
After mixing 5 μl of 7.5 (manufactured by Sigma) on a dropping table, sealing 500 to 1000 μl of the solution having the above-mentioned precipitant composition as a reservoir solution, and then 20 ± 5 ° C. by a vapor diffusion method.
Crystallized. 0.3 × 0.3 × 0.05 mm to 1.2 × 0.6 × 0.
Crystals grew to a size of 05 mm.

Example 14 Preparation of Complex Crystal of Decarbamylase Mutant and DN-Carbamoyl-phenylglycine (II) Known Method (H. Nanba et al., Biosci. Bio)
technology. Biochem. 62, 875-88
Decarbamylase having the amino acid sequence shown in SEQ ID NO: 1 obtained by the method described in No. 1 (1998)) into a decarbamylase solution (0.1 M HEPES buffer, pH 7.5) prepared at a concentration of 15 to 30 mg / ml. A substrate, DN-carbamoyl-phenylglycine, was added, 5 μl of a solution adjusted to a concentration of 5 mM, and 15 to 20% by weight of polyethylene glycol 600 as a precipitant.
0 (manufactured by Nacalai Tesque), 5 μl of 0.1 M HEPES buffer containing 0.2 M lithium chloride, pH 7.5 (manufactured by Sigma) are mixed on a dropping table, and 500 to 1000 μl of a solution having the above precipitant composition is mixed. Was sealed as a reservoir solution, and then crystallized at 20 ± 5 ° C. by a vapor diffusion method. 0.3 × 0.3 in about 2 days to 2 weeks after the start of crystallization
The crystal grew to a size of × 0.05 mm to 1.2 × 0.6 × 0.05 mm.

Example 15 Collection of X-ray Diffraction Data of Complex Crystal with DN-Carbamoyl-Methionine The complex crystal with DN-carbamoyl-methionine prepared in Example 7 was placed in liquid nitrogen. After freezing, R-AXIS is cooled to -170 ° C in a cryo system.
IV ++ (manufactured by Rigaku Corporation), X-ray power 45 kV, 100
Diffraction data measurement was performed using Cu-Kα radiation generated at mA. The diffraction intensity is the crystal-detector distance 120
At an imaging plate (IP) set to 1 mm, 60 frames of diffraction data was collected at 1.8 ° resolution with 1.5 ° oscillation per frame and 3 minutes exposure. The measured diffraction image data is stored in a data processing program (Crystal Clear) attached to R-AXIS.
Using r), the crystallographic parameters were determined and converted to reflection intensity data. The crystallographic parameters are the space group P
2 ₁ 2 ₁ 2, unit cell constant a = 67.4 °, b = 13
7.3 ° and c = 67.9 °.

Example 16 Collection of X-ray Diffraction Data of Complex Crystal with DN-Carbamoyl-valine The complex crystal with DN-carbamoyl-valine prepared in Example 8 was placed in liquid nitrogen. Example 1
Under the conditions and procedures described in 5, crystallographic parameters, 1.8
(4) Reflection intensity data with high resolution was obtained. The crystallographic parameters are the space group P2 ₁ 2 ₁ 2 and the unit cell constant is a = 67.5.
{, B = 137.7} and c = 68.0 °.

Example 17 Collection of X-ray Diffraction Data of Complex Crystal with DN-carbamoyl-phenylalanine The complex crystal with DN-carbamoyl-phenylalanine prepared in Example 9 was placed in liquid nitrogen. After charging and freezing, crystallographic parameters and reflection intensity data at 1.8 ° resolution were obtained under the conditions and procedures described in Example 15. The crystallographic parameters are the space group P2 ₁ 2 ₁ 2 and the unit cell constant is a =
67.6 °, b = 137.5 °, and c = 68.1 °.

[Example 18] Collection of X-ray diffraction data of a complex crystal with DN-carbamoyl-alanine The complex crystal with DN-carbamoyl-alanine prepared in Example 10 was placed in liquid nitrogen. After charging and freezing, crystallographic parameters were obtained under the conditions and procedures described in Example 15.
Reflection intensity data of 2.0 ° resolution was obtained. The crystallographic parameters are as follows: space group P2 ₁ 2 ₁ 2, unit cell constant a = 67.
8 °, b = 137.3 °, and c = 67.9 °.

Example 19 Collection of X-ray Diffraction Data of Complex Crystal with DN-Carbamoyl-parahydroxyphenylglycine DN-carbamoyl-parahydroxyphenylglycine prepared in Example 11 or 13 After the composite crystal was put into liquid nitrogen and frozen, the conditions described in Example 15 were used,
By the procedure, crystallographic parameters and reflection intensity data at 2.0 ° resolution were obtained. The crystallographic parameters are in the space group P2 ₁
2 ₁₂ , unit cell constant a = 67.4 °, b = 137.
4 °, c = 67.9 °.

Example 20 Collection of X-ray Diffraction Data of Complex Crystal with DN-carbamoyl-phenylglycine The complex crystal with DN-carbamoyl-phenylglycine prepared in Example 12 or 14 was collected. After charging and freezing in liquid nitrogen, crystallographic parameters and reflection intensity data at 2.0 ° resolution were obtained under the conditions and procedures described in Example 15. The crystallographic parameters are the space group P2 ₁ 2 ₁ 2, the unit cell constants are a = 67.6 °, b = 137.4 °, and c = 68.
It was 2Å.

Example 21 DN-carbamoyl-
Determination of the three-dimensional structure of the methionine complex The crystal structure of natural type decarbamylase (registration number:) was compared with the crystallographic parameters and reflection intensity data of the DN-carbamoyl-methionine complex crystal obtained in Example 15.
1ERZ) was applied as a reference molecule. In the molecular replacement method, the rotation function and the translation function were calculated using the program CNS, and the orientation and position of the molecule were determined. Using the obtained phase, the electron density of the composite was determined. Next, program O (A. Jones, Uppsala)
(Universitet, Sweden) Display the three-dimensional structural coordinates of the reference molecule, natural decarbamylase, on the electron density diagram displayed on the three-dimensional graphics, correct the model of the site where the amino acid is substituted, and do not fit the electron density Amino acid residue partial structure was modified. Furthermore, a molecular model of DN-carbamoyl-methionine was constructed to match the electron density corresponding to the substrate found near the catalytic residue. The structure refinement program CNS (AT Brunger, Yale) is used as a starting structure with the initial three-dimensional structure model thus constructed.
(University) refinement protocol,
The three-dimensional structure coordinates were refined. In the refinement, the operation of correcting the local structure having a large deviation from the electron density diagram, identifying the electron density corresponding to the water molecule, and including the water molecule in the refinement calculation is repeated, and the R value and the free R value (free R-fac
Using the tor) as an index, the refinement was advanced until the R value became 20% or less. The R value of the final model structure with water molecules added is 6
For reflection data of 9.1-1.8 ° resolution, R value 1
8.3% and a free R value of 21.4%.

Example 22 DN-carbamoyl-
Determination of the three-dimensional structure of the valine complex Using the crystallographic parameters and reflection intensity data of the DN-carbamoyl-valine complex crystal obtained in Example 16, the three-dimensional structure of the complex was obtained according to the procedure described in Example 21. (3D structural coordinates) were determined. The R value of the final model structure to which water molecules were added was 18.8% with respect to the reflection data with a resolution of 69.1-1.8 °.

Example 23 DN-carbamoyl-
Determination of stereostructure of phenylalanine complex Using the crystallographic parameters and reflection intensity data of the DN-carbamoyl-phenylalanine complex crystal obtained in Example 17, according to the procedure described in Example 21, the stereostructure of the complex was determined. (3D structural coordinates) were determined. The R value of the final model structure to which water molecules were added was 18.7% with respect to the reflection data with a resolution of 69.1-1.8 °.

[Example 24] DN-carbamoyl-
Determination of the three-dimensional structure of the alanine complex The three-dimensional structure of the complex according to the procedure described in Example 21 using the crystallographic parameters and reflection intensity data of the DN-carbamoyl-alanine complex crystal obtained in Example 18 (3D structural coordinates) were determined. The R value of the final model structure to which water molecules were added was 18.6% with respect to the reflection data with a resolution of 69.1-1.8 °.

Example 25 DN-carbamoyl-
Stereostructure determination of parahydroxyphenylglycine complex Using the crystallographic parameters and reflection intensity data of the DN-carbamoyl-parahydroxyphenylglycine complex crystal obtained in Example 19, the procedure described in Example 21 was used. The three-dimensional structure (three-dimensional structural coordinates) of the complex was determined. The R value of the final model structure to which water molecules were added was 69.
19.0% for 1-1.8 ° resolution reflection data
Met.

[Example 26] DN-carbamoyl-
Stereostructure determination of phenylglycine complex Using the crystallographic parameters and reflection intensity data of the DN-carbamoyl-phenylglycine complex crystal obtained in Example 20, the procedure of The three-dimensional structure (three-dimensional structural coordinates) was determined. The R value of the final model structure to which water molecules were added was 19.2% with respect to the reflection data with a resolution of 69.1-1.8 °.

[Example 27] Molecular modeling of decarbamylase-substrate complex Three-dimensional structural coordinates of natural type decarbamylase (registration number:
1ERZ), the three-dimensional structural coordinates of the complexes of Examples 21 to 26 were calculated from the Cα atom of 171st cysteine, which is a catalytic residue, using the molecular superposition function of program O (modeling program). Residues within 2.0% (position numbers 43-48, 52, 54, 92-95, 1
09-111, 124-131, 143, 145, 14
9, 156, 167-176, 179-180, 18
3, 191-198, 211, 214-216, 218
-219, 231-236, 244-248), the least-squares superposition operation is performed on the positions of the main chain atoms (N, Cα, C, and O atoms). The three-dimensional structural coordinates of the substrate molecule in the complex are converted into the coordinate system of the natural decarbamylase using the rotation matrix obtained at that time. By inserting the three-dimensional structural coordinates of the converted substrate into the three-dimensional structural coordinates of the natural decarbamylase, an initial structural model of a complex of the substrate and decarbamylase having an activity of hydrolyzing a carbamoyl group was constructed. Using the molecular dynamics calculation program PRESTO (described above), the initial structural model is subjected to optimization of the three-dimensional structural coordinates of the complex by an energy minimization method in which a constraint condition is applied to an atom position other than a hydrogen atom. Was. By applying the above operation to the three-dimensional structure coordinates of all six types of substrate complexes whose structure was determined, the three-dimensional structure coordinates of the complex between the native decarbamylase and the substrate were obtained. In addition, a decarbamylase mutant having the amino acid sequence shown in SEQ ID NO: 2 (H57Y / P203E / V236) is used instead of the three-dimensional structural coordinates of natural decarbamylase.
By using the three-dimensional structural coordinates of (A) (Table 2), the complex of the decarbamylase mutant having a catalytic ability and the substrate can be used.
The dimensional structure coordinates were obtained. FIG. 3 shows the three-dimensional structure around the active site when DN-carbamoyl-methionine was bound to natural decarbamylase to form a complex.

[Example 28] Modification design of pH activity profile and optimality of pH activity of decarbamylase using complex structure Production of D-amino acids by enzymatic method utilizes two kinds of enzymes, hydantoinase and decarbamylase. However, the optimal pH of hydantoinase is around 8-9, and this reaction pH condition is extremely disadvantageous for decarbamylase having an optimal pH near neutrality in terms of enzyme activity and stability. It is. For this reason, production by the enzymatic method is performed in two stages: a hydantoinase reaction and a decarbamylase reaction. If decarbamylase can modify the pH activity profile and optimal pH activity of decarbamylase so that it exhibits the highest enzyme activity and stability in the above pH range, construct an efficient one-step process utilizing two enzymes simultaneously. Becomes possible. In order to modify the optimum pH of decarbamylase, the midpoint of the deprotonation-protonation equilibrium of both Cys171 and Glu46 residues, which is considered to be essential for the catalytic reaction, ie, pK
It is considered that the value a should be shifted to the basic side. 1
One of the design guidelines is to replace the basic amino acid or neutral amino acid around the active site with a neutral amino acid or acidic amino acid and reduce the electrostatic potential around the active center so that the pKa of the catalytic residue Cys171 becomes basic. A way to shift to the side was considered. The selection of the amino acid mutation site
Calculation of electrostatic potential (Takahashi et al., (1992), Bio
Polymers 32, 897-909), residues that greatly influence the electrostatic potential formed by Cys171, which is a catalytic residue, were identified by the following procedure. (1) To the sulfur atom (Sγ) of Cys171 side chain
Giving a charge of 1.0, the decarbamylase dimer (molecule A
And the charge of all other atoms of the molecule B) is zero,
Calculate the electrostatic potential that creates a -1.0 charge on the Sγ atom of Cys171. (2) From the obtained electrostatic potential, calculate the electrostatic contribution of all amino acid residues other than Cys171 to Cys171. Table 9 shows amino acid residues having a large effect on the electrostatic potential of the catalytic residue Cys171, their potentials, and the distance between Cα atoms.

Of the residues that greatly affect the electrostatic potential of the catalytic residues, Lys126, Glu46, Gl46
Since u145, Arg174 and Arg175 are residues involved in catalytic activity and / or substrate binding, these sites were excluded from mutation candidates. From the calculation results, Lys235, Arg55, Arg13
9, Asn196 was selected. For each part, check the local structure and interaction around it, and use the program SHRIKE
The validity of the structural change due to the mutation was evaluated using (described above), and the following mutation was designed.

Position 235 K235Q or K235
E, position 55 R55I, R55L, or R55E;
Positions 139 R139N, 196 N196D In this design, one amino acid mutation at each position and 2
Multiple mutants combining multiple mutations are designed. Note that the program PRESTO or the program AMBER can be used instead of the program SHRIKE.

[0117]

[Table 9]

[0118]

The three-dimensional structural coordinates of decarbamylase and the complex of decarbamylase and the substrate provided by the present inventors are not merely data for defining the structure of the substance, but are various data other than the substance. By processing and processing the information of the three-dimensional structure coordinates itself using hardware resources by a design program, stability such as heat resistance, organic solvent resistance, resistance to air oxidation, etc., particularly, pH activity profile of enzyme reaction The present invention is a useful "thing" invention capable of molecularly designing amino acid mutations relating to a change in pH activity optimality, a change in specific activity and a change in substrate specificity. Thus, for example, as shown in Example 28, a modified enzyme advantageous for industrial use can be quickly and efficiently obtained.

[0119]

[Sequence List] SEQUENCE LISTING <110> Kaneka Corporation <120> Three-dimensional structure of decarbamylase complex <130> P01-0171 <140> ND <141> 2001-02-28 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 303 <212> PRT <213> E coli. <400> 1 Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala Arg 1 5 10 15 Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Asp Met Leu Thr 20 25 30 Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu Ala 35 40 45 Leu Thr Thr Phe Phe Pro Arg Trp His Phe Thr Asp Glu Ala Glu Leu 50 55 60 Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro Leu 65 70 75 80 Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr Ala 85 90 95 Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser Ile 100 105 110 Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile His 115 120 125 Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His Leu 130 135 140 Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr Asp 145 150 155 160 Val Asp Al a Ala Lys Met Gly Met Phe Ile Ala Asn Asp Arg Arg Trp 165 170 175 Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile Cys 180 185 190 Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His Asp 195 200 205 His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser Tyr 210 215 220 Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Ala Gly Met Glu Glu 225 230 235 240 Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly Glu 245 250 255 Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala Ala 260 265 270 Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn Phe 275 280 285 Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu 290 295 300 <210> 2 <211> 303 <212> PRT <213> E.coli <400> 2 Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala Arg 1 5 10 15 Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Asp Met Leu Thr 20 25 30 Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu Ala 35 40 45 Leu Thr Thr Phe Phe Pro Arg Trp Tyr Phe Thr Asp Glu Ala G lu Leu 50 55 60 Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro Leu 65 70 75 80 Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr Ala 85 90 95 Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser Ile 100 105 110 Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile His 115 120 125 Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His Leu 130 135 140 Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr Asp 145 150 155 160 Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg Trp 165 170 175 Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile Cys 180 185 190 Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Glu Val Pro Gln His Asp 195 200 205 His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser Tyr 210 215 220 Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Ala Gly Met Glu Glu 225 230 235 240 Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly Glu 245 250 255 Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala Ala 260 265 270 Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn Phe 275 280 285 Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu 290 295 300 <210> 3 <211> 303 <212> PRT <213> E coli. <400> 3 Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala Arg 1 5 10 15 Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Asp Met Leu Thr 20 25 30 Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu Ala 35 40 45 Leu Thr Thr Phe Phe Pro Arg Trp His Phe Thr Asp Glu Ala Glu Leu 50 55 60 Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro Leu 65 70 75 80 Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr Ala 85 90 95 Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser Ile 100 105 110 Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile His 115 120 125 Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His Leu 130 135 140 Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr Asp 145 150 155 160 Val Asp Ala Ala Lys Met Gly Met Phe Ile Cy s Asn Asp Arg Arg Trp 165 170 175 Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile Cys 180 185 190 Gly Gly Tly Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His Asp 195 200 205 His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser Tyr 210 215 220 Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Val Gly Met Glu Glu 225 230 235 240 Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly Glu 245 250 255 Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala Ala 260 265 270 Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn Phe 275 280 285 Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu 290 295 300

[Brief description of the drawings]

FIG. 1 is a ribbon diagram showing the main chain of decarbamylase in a complex of natural decarbamylase and DN-carbamoyl-methionine. Cys171 (natural type), Ala171
(Complex) and the substrate are ball-and-stick
Shown by model. N indicates the N-terminus of the enzyme, and C indicates the C-terminus of the enzyme.

FIG. 2 is a schematic diagram showing a substrate binding site of a decarbamylase complex.

FIG. 3 is a schematic diagram showing a three-dimensional structure model of a substrate binding site of a complex of natural decarbamylase and DN-carbamoyl-methionine.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Soichi Morikawa 46-5 Kawamacho, Himeji-shi, Hyogo Meiji Grand Building Shirasagi Room 302 F-term (reference) 4B024 AA11 BA11 CA03 CA06 GA25 HA01 4B050 CC03 CC04 DD02 LL03

Claims (69)

[Claims] 1. A crystal of decarbamylase having a moiety that binds DN-carbamoyl-α-amino acid, or a mutant thereof. 2. The crystal according to claim 1, wherein the decarbamylase or a mutant thereof has the amino acid sequence shown in SEQ ID NO: 1. 3. The crystal according to claim 1, wherein the decarbamylase or a mutant thereof has the amino acid sequence shown in SEQ ID NO: 2. 4. The crystallographic symmetry is in the space group P2 ₁ 2 ₁ 2 and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136.
The crystal according to claim 2, having a size of ± 10 ° and c = 68 ± 5 °. 5. The crystallographic symmetry is space group P2 ₁ 2 ₁ 2, the unit lattice constant of the crystal a = 67 ± 5Å, b = 136
The crystal according to claim 3, having a size of ± 10 ° and c = 68 ± 5 °. 6. The use of the crystal according to claim 1, which is used for obtaining three-dimensional structural coordinates of a decarbamylase having a moiety that binds DN-carbamoyl-α-amino acid, or a mutant thereof. 7. The enzyme substrate specificity, catalytic activity, stability,
identifying, searching for one or more properties selected from the group consisting of pH activity profile, pH activity optimality, and water solubility;
A three-dimensional structural coordinate of decarbamylase having a portion that binds DN-carbamoyl-α-amino acid, or a mutant thereof, used for evaluation, change, or design. 8. The three-dimensional structural coordinates according to claim 7, wherein the decarbamylase or a mutant thereof has the amino acid sequence shown in SEQ ID NO: 1, and the three-dimensional structural coordinates are shown in Table 1. 9. The three-dimensional structural coordinates according to claim 7, wherein the decarbamylase or a mutant thereof has the amino acid sequence shown in SEQ ID NO: 2, and the three-dimensional structural coordinates are those shown in Table 2. 10. A portion which binds to DN-carbamoyl-α-amino acid, wherein the structural coordinate homology RMSD with the three-dimensional structural coordinate according to claim 8 or 9 is 3.0 ° or less. Three-dimensional structural coordinates of decarbamylase or a variant thereof. 11. A crystal of a complex of a decarbamylase having a portion that binds DN-carbamoyl-α-amino acid, or a mutant thereof, and DN-carbamoyl-α-amino acid. 12. The method according to claim 11, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
-N-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN
Carbamoyl-phenylalanine, DN-carbamoyl-glycine, DN-carbamoyl-histidine,
DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN-carbamoyl-leucine, D
-N-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-proline,
DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine, D
-N-carbamoyl-threonine, DN-carbamoyl-valine, DN-carbamoyl-tryptophan,
12. The complex of claim 11, wherein the complex is selected from the group consisting of and DN-carbamoyl-tyrosine. 13. A decarbamylase having a moiety that binds DN-carbamoyl-α-amino acid, or a mutant thereof, removes the carbamoyl group that modifies DN-carbamoyl-α-amino acid to remove D-α-amino acid. -Crystals of the complex according to claims 11 and 12, which have no activity of converting into amino acids. 14. The complex crystal according to claim 13, wherein the decarbamylase or a variant thereof has the amino acid sequence shown in SEQ ID NO: 1. 15. The DN-carbamoyl-α-amino acid is DN-carbamoyl-methionine, the crystallographic symmetry is a space group P2 ₁ 2 ₁ 2 and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136 ± 10 °, c = 68 ±
The composite crystal according to claim 14, having a size of 5 °. 16. The DN-carbamoyl-α-amino acid is DN-carbamoyl-valine, the crystallographic symmetry is a space group P2 ₁ 2 ₁ 2 and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136 ± 10 °, c = 68 ± 5 °
The composite crystal according to claim 14, having a size of: 17. The method of claim 17, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylalanine, the crystallographic symmetry is the space group P2 ₁ 2 ₁ 2, and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136 ± 10 °, c
The crystal of the composite according to claim 14, having a size of = 68 ± 5 °. 18. The DN-carbamoyl-α-amino acid is DN-carbamoyl-alanine, the crystallographic symmetry is a space group P2 ₁ 2 ₁ 2 and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136 ± 10 °, c = 68 ± 5
The composite crystal according to claim 14, having a size of Å. 19. A DN-carbamoyl-α-amino acid
Is DN-carbamoyl-parahydroxyphenyl
Lysine whose crystallographic symmetry is in the space group P2 ₁2₁2
And the unit cell constant of the crystal is a = 67 ± 5 °, b = 136
15. The apparatus according to claim 14, which has a size of ± 10 ° and c = 68 ± 5 °.
A crystal of the complex described. 20. The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine, the crystallographic symmetry of which is space group P2 ₁ 2 ₁ 2, and the unit cell constant of the crystal is a = 67 ± 5 °, b = 136 ± 10 °, c
The crystal of the composite according to claim 14, having a size of = 68 ± 5 °. 21. A complex in which a stoichiometrically equivalent amount of DN-carbamoyl-α-amino acid and a decarbamylase having a portion that binds DN-carbamoyl-α-amino acid, or a mutant thereof are combined. 21. The crystal of the complex according to claim 11, wherein the complex further comprises two molecules in the crystallographic asymmetric unit to form an aggregate. 22. A method for obtaining three-dimensional structural coordinates of a complex of decarbamylase having a portion binding to DN-carbamoyl-α-amino acid or a mutant thereof and DN-carbamoyl-α-amino acid. Claim 11.
22. Use of a crystal of the complex according to any one of to 21. 23. Identify, search, evaluate, modify or design one or more properties selected from the group consisting of enzyme substrate specificity, catalytic activity, stability, pH activity profile, pH activity optimum, and water solubility. To use, DN-
Carbamoyl-α-amino acid and DN-carbamoyl-
Three-dimensional structural coordinates of a complex with a decarbamylase having a portion that binds an α-amino acid, or a mutant thereof. 24. The method according to claim 24, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
-N-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN
Carbamoyl-phenylalanine, DN-carbamoyl-glycine, DN-carbamoyl-histidine,
DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN-carbamoyl-leucine, D
-N-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-proline,
DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine, D
-N-carbamoyl-threonine, DN-carbamoyl-valine, DN-carbamoyl-tryptophan,
The three-dimensional structural coordinate of the complex according to claim 23, wherein the three-dimensional structural coordinate is selected from the group consisting of and DN-carbamoyl-tyrosine. 25. A decarbamylase having a portion that binds DN-carbamoyl-α-amino acid and DN-carbamoyl-α-amino acid, or a variant thereof is DN
24. A complex with a DN-carbamoyl-α-amino acid according to claims 22 and 23, which has no activity of removing the carbamoyl group modifying the carbamoyl-α-amino acid and converting it to a D-α-amino acid. Three-dimensional structural coordinates. 26. The decarbamylase or a variant thereof has the amino acid sequence shown in SEQ ID NO: 1.
6. A three-dimensional structural coordinate of the complex with the DN-carbamoyl-α-amino acid according to 5. 27. The three-dimensional structure of the complex according to claim 26, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-methionine, and the three-dimensional structural coordinates are shown in Table 3. Structure coordinates. 28. The three-dimensional structure of the conjugate according to claim 26, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-valine and the three-dimensional structural coordinates are shown in Table 4. Structure coordinates. 29. The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylalanine,
27. The three-dimensional structural coordinates are as shown in Table 5.
The three-dimensional structural coordinates of the complex according to 1. 30. The three-dimensional structure of the complex according to claim 26, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-alanine, and the three-dimensional structural coordinates are shown in Table 6. Structure coordinates. 31. The complex according to claim 26, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-parahydroxyphenylglycine and the three-dimensional structural coordinates are as shown in Table 7. Three-dimensional structural coordinates. 32. The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
27. The three-dimensional structural coordinates are as shown in Table 8.
The three-dimensional structural coordinates of the complex according to 1. 33. The complex 3 according to claim 27, wherein
Coordinate homology RMS with three-dimensional structure coordinates of decarbamylase or a mutant thereof contained in three-dimensional structure coordinates
A three-dimensional structural coordinate of decarbamylase having a portion binding to DN-carbamoyl-α-amino acid, or a mutant thereof, wherein D is 3.0 ° or less. 34. The complex 3 according to claim 27, wherein
From the three-dimensional structural coordinates of the decarbamylase having a portion that binds DN-carbamoyl-α-amino acid according to claim 9 or the three-dimensional structural coordinates of a decarbamylase having the amino acid sequence shown in SEQ ID NO: 3,
3D structural coordinates of the complex constructed by the molecular design method. 35. Identify, search, evaluate, modify or design one or more properties selected from the group consisting of enzyme substrate specificity, catalytic activity, stability, pH activity profile, pH activity optimum, and water solubility. 35. A recording medium storing all or a part of the three-dimensional structure coordinates according to any one of claims 8, 9, 27 to 32 and 34, and use thereof. 36. All or one of the three-dimensional structural coordinates according to any one of claims 8, 9, and 27 to 32 in X-ray crystal structure analysis of a protein and a protein complex using a molecular replacement method. Recording medium storing the unit and its use. 37. A method for designing a decarbamylase mutant, which comprises modifying a physical property or a function based on the three-dimensional structural coordinates according to any one of claims 7 to 10 and 23 to 34. A method comprising the step of designing. 38. A method for designing a decarbamylase mutant, comprising the steps of: generating a crystal of an enzyme having decarbamylase activity; determining the three-dimensional structural coordinates of the crystal by X-ray crystal structure analysis of the crystal; 3 decided
A method comprising designing a decarbamylase mutant having improved properties or functions based on the dimensional structure coordinates. 39. The method according to claim 38, wherein the three-dimensional structure coordinates are the three-dimensional structure coordinates according to any one of claims 7 to 10 and 23 to 34. 40. A method for producing a mutant decarbamylase, comprising the steps of: producing a crystal of an enzyme having decarbamylase activity; determining the three-dimensional structural coordinates of the crystal by X-ray crystal structure analysis of the crystal. A method of designing a decarbamylase variant having improved properties or functions based on the obtained three-dimensional structural coordinates, and a step of producing the decarbamylase variant. 41. The method according to claim 40, wherein the three-dimensional structure coordinates are the three-dimensional structure coordinates according to any one of claims 7 to 10 and 23 to 34. 42. The step of designing a decarbamylase variant comprises modifying one or more enzymatic properties selected from the group consisting of substrate specificity, catalytic activity, stability, pH activity profile, pH activity optimality and water solubility. 42. A method according to claim 41 for the purpose of: 43. The method of claim 42, wherein altering the enzymatic properties comprises improving stability. 44. The method of claim 42, wherein altering the enzymatic properties comprises altering catalytic activity and optimizing pH activity profile, pH activity optimality. 45. The method according to claim 45, wherein the modification of the enzyme properties is within 12%,
More preferably, at one or more amino acid residue positions at a distance from the substrate binding site within 8 °, even more preferably within 5 °, or at amino acid residue positions exposed on the surface of the enzyme. By substituting, deleting or inserting amino acid residues, the pH activity profile of decarbamylase, or a mutant thereof,
46. The method of claim 44, wherein the activity optimality, pH stability profile, or pH stability optimality is altered. 46. A decarbamylase mutant obtained by the production method according to any one of claims 40 to 45. 47. A decarbamylase having the three-dimensional structural coordinates according to claim 33 or 34, or a mutant thereof. 48. A method for producing a D-α-amino acid using DN-carbamoyl-α-amino acid as a raw material, comprising using the decarbamylase according to claim 47 or a mutant thereof as a catalytic enzyme. 49. A method for modifying the optimum pH of the decarbamylase shown in SEQ ID NO: 3, which is a midpoint of the equilibrium between deprotonation and protonation of both Cys171 and Glu46 residues, which is considered to be essential for the catalytic reaction. In order to shift the value to the basic side, basic amino acids and neutral amino acids around the active site are replaced with neutral amino acids and acidic amino acids, and the electrostatic potential around the active center is lowered, so that it is a catalytic residue. This is a method in which the pKa of Cys171 is shifted to the basic side. (1) A charge of -1.0 is given to a sulfur atom (Sγ) of a Cys171 side chain, and decarbamylase dimer (molecule A and molecule B) With the charges of all other atoms being zero, calculate the electrostatic potential that creates a -1.0 charge on the Sγ atom of Cys171,
(2) By calculating the electrostatic contribution of all amino acid residues other than Cys171 to Cys171 from the obtained electrostatic potential, residues that greatly affect the electrostatic potential are identified, and the identified residue is identified. Investigate the local structure and interaction of the group, and program SHRIKE,
A method by calculating the validity of the structural change of a mutation at each residue by the program PRESTO or the program AMBER. 50. A decarbamylase having the amino acid sequence represented by SEQ ID NO: 3, and further comprising K235Q, K
235E, R55I, R55L, R55E, R139
A mutant having at least one mutation of N and N196D. 51. has the amino acid sequence of SEQ ID NO: 1,
A decarbamylase having the three-dimensional structure coordinates shown in Table 1. 52. It has the amino acid sequence of SEQ ID NO: 2,
A decarbamylase whose three-dimensional structure coordinates have the three-dimensional structure coordinates shown in Table 2. 53. The structure coordinate homology RMS of the decarbamylase according to claim 51 or 52 with the three-dimensional structure coordinates.
A decarbamylase having a D-N-carbamoyl-α-amino acid-binding portion or a variant thereof, wherein D is 3.0 ° or less. 54. A complex of a DN-carbamoyl-α-amino acid and a decarbamylase or a mutant thereof having a portion that binds DN-carbamoyl-α-amino acid, wherein the specific three-dimensional structural coordinate is Complex having. 55. The method wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
-N-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine, DN-carbamoyl-aspartic acid, DN-carbamoyl-glutamic acid, DN
Carbamoyl-phenylalanine, DN-carbamoyl-glycine, DN-carbamoyl-histidine,
DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN-carbamoyl-leucine, D
-N-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-proline,
DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine, D
-N-carbamoyl-threonine, DN-carbamoyl-valine, DN-carbamoyl-tryptophan,
And a conjugate having particular three-dimensional structural coordinates selected from the group consisting of: DN-carbamoyl-tyrosine. 56. The complex according to claim 54, wherein the decarbamylase or a mutant thereof has the amino acid sequence specified by SEQ ID NO: 1. 57. The complex according to claim 56, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-methionine and the three-dimensional structural coordinates are as shown in Table 3. 58. The complex according to claim 56, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-valine and the three-dimensional structural coordinates are as shown in Table 4. 59. The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylalanine,
57. The three-dimensional structural coordinates are as shown in Table 5.
The complex according to any one of the above. 60. The complex according to claim 56, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-alanine and the three-dimensional structural coordinates are as shown in Table 6. 61. The complex according to claim 56, wherein the DN-carbamoyl-α-amino acid is DN-carbamoyl-parahydroxyphenylglycine and the three-dimensional structural coordinates are as shown in Table 7. . 62. The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine,
57. The three-dimensional structural coordinates are as shown in Table 8.
The complex according to any one of the above. 63. The complex 3 according to claim 54, wherein
D, wherein the structural coordinate homology RMSD with the three-dimensional structural coordinate of the complex of decarbamylase or a mutant thereof contained in the three-dimensional structural coordinates is 3.0 ° or less,
A complex with a decarbamylase having a moiety that binds -N-carbamoyl-α-amino acid, or a mutant thereof. 64. The complex 3 according to claim 54, wherein
From the three-dimensional structural coordinates of the decarbamylase having a portion that binds DN-carbamoyl-α-amino acid according to claim 9 or the three-dimensional structural coordinates of a decarbamylase having the amino acid sequence shown in SEQ ID NO: 3,
Complex with three-dimensional structural coordinates constructed by molecular design techniques. 65. An enzyme molecule having a decarbamylase activity using a DN-carbamoyl-α-amino acid as a substrate, and a decarbamylase mutant having at least one or more of the following characteristics in substrate binding: (1) Asn172 Side chain amide group, Arg174, Ar
The side chain guanidino group of g175 and the side chain hydroxyl group of Thr197 can form an ionic bond or a hydrogen bond with the α-carboxyl group of the substrate; (2) the main chain carbonyl group of Asn196 forms an α-amide group and hydrogen of the substrate (3) the side chain amino group of Lys126 and the main chain amide group of Asn172 can form a hydrogen bond with the carbonyl group of carbamoyl substrate; (4) the main chain carbonyl group of Asn196 and Glu46 The side chain carboxyl group can form a hydrogen bond with the amide group of the carbamoyl substrate; or (5) Pro130, His143, Pro198 and Ile285 of one of the enzyme molecules forming a dimer.
Can interact hydrophobically with the D-amino acid side chain of the substrate. 66. A decarbamylase variant having the amino acid sequence specified by SEQ ID NO: 1. 67. A method for determining the three-dimensional structural coordinates of a complex of a decarbamylase or a mutant thereof and a substrate, comprising the steps of:
A complex of a decarbamylase or a mutant thereof with a substrate, using a crystal of a complex of a substrate and a mutant having an activity of binding to carbamoyl-α-amino acids but not having an activity of hydrolyzing an N-carbamoyl group; How to determine dimensional structure coordinates. 68. The method of determining a three-dimensional structure coordinate of a complex of the decarbamylase or a mutant thereof according to claim 67 and a substrate, wherein the N-carbamoyl has a binding activity with DN-carbamoyl-α-amino acids. The three-dimensional structural coordinates of the decarbamylase mutant complex are determined using a complex crystal of the mutant having no activity of hydrolyzing the group and the substrate, and the three-dimensional structural coordinates are compared with the natural decarbamylase or N-carbamoyl group. A step of superimposing the three-dimensional structural coordinates of the decarbamylase mutant having the activity of hydrolyzing the compound with the substrate and the decarbamylase mutant having the activity of hydrolyzing the N-carbamoyl group and the substrate. How to determine dimensional structure coordinates. 69. The method for determining a three-dimensional structure coordinate of a complex of a carbamylase and a substrate according to claim 67 or 68, wherein the mutant according to claim 66 is used.