JP3124967B2 - Enzymes used in the production of tetrahydrobiopterin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing 6- (R) -erythro-5,6,7,8-tetrahydrobiopterin (hereinafter sometimes referred to as BH4).

More particularly, it relates to an improved method for producing BH4 by an enzymatic method.

[0003]

2. Description of the Related Art Tetrahydrobiopterin has, for example, D-type by the relative configuration of the side chain of the carbon atom at the 6-position.
There are a series and an L-series, and the tetrahydro form has (R)-according to the absolute configuration at the carbon atom at the 6-position.
It is known that there are two diastereomers, or (S)-. That is, the general formula (II):

[0004]

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[0005] In the tetrahydropterin derivative represented by the formula:

[0006]

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Is BH4. BH4 is present in vivo, is a coenzyme of monooxygenase involved in monoamine neurotransmitter synthesis, and is considered to be one of its regulators. Thus, a deficiency in BH4 results in a deficiency of the neurotransmitters serotonin, dopamine, noradrenaline, adrenaline, etc., leading to severe neurological symptoms. For example, malignant phenylketonuria is known, and in fact, pioneering treatment results by 6- (R, S) -tetrahydrobiopterin administration have been reported (Niederwieser, A. et al., Lancet, 1, 550). (1979);
Curtius, H-CH et al., Clin. Chim. Acta, 98 , 251-262 (19
79)).

[0008] As a method for producing tetrahydrobiopterin, a method for chemically or enzymatically reducing L-erythro-biopterin is known.

However, according to the chemical method, the synthesis of biopterin itself is expensive and complicated, and 6R
A mixture of the isomer and the 6S isomer, which must be resolved, but this resolution is extremely difficult, and its advantageous resolution is not known at present, and studies have been made to increase the yield of the 6R isomer. ing. On the other hand, the enzymatic method has an advantage that only the 6R form can be obtained, but has a problem that the apparatus and the operation are complicated.

Have reported a method using BH4 enzymatic method starting from guanosine 5'-triphosphate (GTP) present in various animal tissues and yeast (BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICAT).
ION, 128 , No. 3, 1093-1107 (1985)). The method consists of the following scheme.

[0011]

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[0012]

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However, as is clear from the above scheme, the method of Milestein et al. Requires a complicated operation comprising a plurality of reaction steps, and requires an intermediate product, especially 6
-Pyrvoyl-tetrahydropterin is very unstable, and it is difficult to react it with sepiapterin reductase after the separation and the final reaction step, and it cannot be said to be a very practical method. In addition, since no single purified enzyme is used in all the steps, it may be difficult to separate BH4 from by-products (including unidentified products).

[0014]

The inventors of the present invention have conducted intensive studies in view of the above situation, and as a result, have found an enzyme system which can efficiently produce BH4 in one pot using GTP as a starting material. It was completed.

[0015]

That is, the present invention provides a GTP
Reacting with Escherichia coli-derived GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase to formula (I):

[0016]

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6- (R) -L-erythro-
A method for producing 5,6,7,8-tetrahydrobiopterin is provided.

The present invention relates to an enzyme used in the above-mentioned production method, wherein (1) the amino acid sequence from the amino terminal to the 25th amino acid has the formula
Pro-Ser-Leu-Ser-Lys-Glu-Ala-Ala-Leu-Val-His-Glu-Al
a-Leu-Val-Ala-Arg-Gly-Leu-Glu-Thr-Pro-Leu-Arg-Pro, having the ability to convert GTP to D-erythro-7,8-dihydroneopterin triphosphate GTP cyclohydrolase I derived from Escherichia coli, (2) The amino acid sequence from the amino terminal to the 15th is represented by the formula: Val-Gly-Leu-Arg-Arg-Arg-Ala-Arg-Leu-Ser-Arg-Leu-Va
1-Ser-Phe and D-erythro-7,8-dihydroneopterin triphosphate was added to 6-pyruvoyl-5,6,7,8
-Rat liver-derived 6-pyruvoyltetrahydropterin synthase capable of converting to tetrahydropterin,
And (3) the partial amino acid sequence has the formula: Leu-Leu-Ser-Leu-Leu-Gln-Arg-Asp-Thr-Phe-Gln-Ser-Gl
y-Ala-His-Val-Asp-Phe-Tyr-Asp-Ile, and 6-pyruvoyl-5,6,7,8-tetrahydropterin with 6- (R) -L-erythro-5,6, 7,
Also provided is rat erythrocyte-derived sepiapterin reductase which has the ability to convert to 8-tetrahydrobiopterin.

GT used as a starting material in the present invention
P can be obtained by separating and purifying P from acid extracts of various animal tissues and yeast according to a conventional method. In addition, commercially available G
TP may be used.

The enzyme system of the present invention can be obtained from E. coli-derived GTP cyclohydrolase I (hereinafter, referred to as GCH), for example, 6-pyruvoyltetrahydropterin synthase (hereinafter, referred to as PTPS) obtained from rat liver and, for example, obtained from rat erythrocytes. Sepiapterin reductase (hereinafter S
PR). These enzymes are preferably used as a mixture in a solution state, but may be used alone or together and immobilized by a usual method, for example, a carrier binding method or an entrapping method.

In the present invention, the above three enzymes are BH4
Can be used at a concentration sufficient for the production of the enzyme. According to the present invention, all of these enzymes can be used for SDS electrophoresis or HPL.
Also provided is a method of purifying into a substantially pure single purified preparation by analysis of C. If all enzymes are used as a single purified sample,
Is expected to minimize the formation of by-products of unknown structure,
Separation and recovery of BH4 is also easy.

In the enzyme system of the present invention, it is important that GTP cyclohydrolase I (GCH) is derived from Escherichia coli. When GCH is substituted by a source other than Escherichia coli, for example, GCH derived from a rat, the enzyme is converted to the final product B
Inhibited by H4 (30% inhibited by 10 μM of BH4), and furthermore, 6-pyruvoyl-5,6,7, of D-erythro-7,8-dihydroneopterin triphosphate.
MgC required for conversion to 8-tetrahydropterin
The enzyme system containing the rat-derived enzyme cannot be used for a one-pot reaction as in the production method of the present invention because the enzyme is also inhibited by l ₂ (50% inhibition with 0.1 mM of MgCl ₂ ).

In the production method of the present invention, GCH in the reaction system
The concentration is preferably 500-5000 μg / ml, particularly preferably 5000 μg / ml, and the PTPS concentration is preferably 5 μg / ml.
0-500 μg / ml, particularly preferably 500 μg / ml,
The SPR concentration is preferably 4 to 40 µg / ml, particularly preferably 40 µg / ml. However, the upper limit of the amount of the enzyme exemplified here is mainly determined by the final concentration of the enzyme in the purified sample, and there is no substantial upper limit in the amount of the enzyme as long as the enzyme activity is stable.

GCH used in the production method of the present invention is derived from Escherichia coli, and can be obtained by lysis of Escherichia coli such as strain B (wild strain), ammonium sulfate fractionation, AcA34 gel filtration, and GTP-sepharose fractionation. The B strain is, for example, JE603
E. coli or A available as 4 (National Institute of Genetics)
E. coli available as TCC 23848.

PTPS is obtained by subjecting a crude extract obtained from the liver of a mammal such as a rat to ammonium sulfate fractionation, heat treatment, hydroxyapatite chromatography, butyl toyopearl chromatography, Sephadex G100 gel filtration, Mo
It can be obtained by purification by no Q ion exchange chromatography.

The SPR can be obtained by hemolysis of erythrocytes of mammals such as rats, ammonium sulfate fractionation, butyl toyopearl chromatography, hydroxyapatite chromatography, and Red Sepharose CL-6B.

[0027] The enzyme preparation obtained in this way is SD
By S-polyacrylamide gel electrophoresis, it is possible to know the degree of purification or whether it has been sufficiently purified and isolated, and if necessary, the molecular weight. Further, the amino acid sequence at the amino terminus of the obtained enzyme preparation is analyzed by an automatic amino acid sequence analyzer. Alternatively, the obtained enzyme preparation is decomposed using a protease, the peptide is separated using high performance liquid chromatography, and the amino acid sequence can be similarly determined.

In the present invention, as described above, enzymes which are particularly suitable for use in the production method of the present invention are also provided. However, each of these enzymes has an amino acid sequence having a new structure due to its amino terminal amino acid sequence and partial amino acid sequence. It turned out to be. The present invention provides a material for mass production of the enzyme of the present invention by isolating the gene of the present enzyme, which encodes the novel amino acid sequences, and also by using recombinant DNA technology, by discovering new amino acid sequences.

In the production method of the present invention, GCH is KCl
In activated, it PTPS the Mg ^{2 +} is essential for expression of activity, also in consideration of the fact that require NADPH in the active expression of the SPR, these activators and coenzymes will not inhibit each other other enzymatic reactions What is necessary is just to set in a density range. The concentration of KCl is preferably 0.05 to 0.50.
M, particularly preferably a 0.10 M, MgCl ₂ concentration of the Mg ^{2 +} sources is preferably 5 to 50
The concentration of mM, particularly preferably 5-20 mM, and NADPH is preferably 4 mM or more, particularly preferably 4 mM. The concentration of the substrate GTP is not particularly limited, but may be 0.2 mM
The following is preferable, and 0.1 to 0.2 mM is particularly preferable. G
When the concentration of TP greatly exceeds 0.2 mM, there is a disadvantage that the conversion efficiency of GTP to dihydroneopterin triphosphate is reduced.

Various reaction conditions may be set within a range that does not inhibit the reaction. As a solvent, a reducing agent such as dithioerythritol or dithiothreitol or a heavy metal chelator such as ethylenediaminetetraacetic acid (EDTA) may be appropriately added. , A suitable pH regulator such as a phosphate buffer may be used, and the reaction pH and temperature are determined by the characteristics of the present enzyme. The pH is preferably about 6 to 8, particularly preferably about 7, and the temperature is about 25 to 42 ° C. Particularly, the temperature is preferably around 37 ° C. The reaction time is generally from 5 minutes to 1 hour. The BH4 thus generated in the reaction mixture can be recovered by a separation method using chromatography such as HPLC or the like. The yield based on the raw material (GTP) of BH4 according to the process of the present invention is:
It is about 80% or more, and the yield is extremely high as compared with the conventional method.

As described above, the rat-derived GT
P-cyclohydrolase I is a rate-limiting enzyme for BH4 synthesis and is known to be subject to various activities.
Therefore, the present inventors have purified GTP cyclohydrolase I from rat liver and clarified its properties. CDNA cloning of this enzyme was performed, and the entire primary structure was determined.
A λZAPII cDNA library was prepared from rat liver poly (A) ⁺ RNA, an oligonucleotide was synthesized based on the partial amino acid sequence of the peptide obtained by lysyl endopeptidase digestion, and screening was performed using the oligonucleotide as a probe. A clone having the inserted fragment was obtained. This clone encoded a polypeptide consisting of 241 amino acid residues, and contained eight regions completely matching the amino acid sequences of the eight peptides separated by the lysyl endopeptidase digestion. Comparison of the amino acid sequence predicted from the nucleotide sequence with the NBRF protein database shows that a part of the amino acid sequence of GTP cyclohydrolase I shows homology with the amino acid sequence identified as the dihydrofolate binding site of dihydrofolate reductase. Was revealed. Since the activity of this enzyme is inhibited by BH4 which is similar to the structure of dihydrofolate, it is considered that this part is a pterin binding site.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the examples.

[0033]

Example 1 Extraction and Purification of GCH As Escherichia coli, strain B (wild strain, National Institute of Genetics, JE6)
034) was used. The E. coli B strain was lysed, fractionated with ammonium sulfate, filtered with AcA34 gel, and fractionated with GTP-Sepharose as follows, and purified about 1400-fold. The amount of protein, activity yield and specific activity in each step are shown in Table 1 below.

Lysis procedure : 245 g of Escherichia coli stored at -70 ° C. were mixed with 420 ml of 0.3 M Tris-HCl buffer (buffer A), pH 8.0 in a blender, and mixed for 5 times each.
Sonication for 5 minutes was performed 5 times to destroy the bacteria. This lysate was centrifuged at 12,000 × g for 20 minutes to obtain a supernatant.
The precipitate was suspended in 91 ml of buffer A, sonicated for 5 minutes and centrifuged in the same manner to obtain a supernatant, which was mixed with the above supernatant.

Ammonium sulfate fractionation : After adding 229 ml of 100% saturated ammonium sulfate to 535 ml of the lysate and stirring for 20 minutes, 12,000 × g
For 20 minutes to obtain a supernatant. 722 ml of this supernatant
149 ml of 100% saturated ammonium sulfate was added to the mixture and stirred for 20 minutes.
Centrifugation was performed at 12,000 × g for 20 minutes to obtain a precipitate. The precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 0.3 M KCl (buffer B), and dialyzed against 5 L of buffer B by changing twice.

AcA34 gel filtration : dialyzed enzyme solution
After centrifugation at 2000 × g for 20 minutes, a supernatant was obtained. An ultrafiltration membrane (PM-10, manufactured by Amicon) is used for 57 ml of the supernatant.
And concentrated to 24 ml. The concentrated enzyme solution was subjected to gel filtration at a flow rate of 60 ml / hour using a 5 × 83 cm AcA34 gel column equilibrated with buffer B to obtain an active fraction.

GTP-Sepharose fractionation : The active fraction of the gel filtration was converted to 10 mM pH 7.0 containing 2.5 mM EDTA.
GTP equilibrated with potassium phosphate buffer (buffer C)
-Adsorbed on a Sepharose column at a flow rate of 60 ml / hour,
40 ml of buffer C containing 0.3 M KCl and buffer C90
Washed successively with ml. Next, 0.25 mg /
flow buffer C containing 10 ml of GTP at a flow rate of 10 ml / hour,
An active fraction of GCH was obtained.

[0038]

[Table 1] Table 1 -------------------------------------------- ---------- Process protein activity Specific activity (nmol / h content (mg) yield (%) / mg protein) ------------------ ------------------------------------ Lysis 33,000 100 0.25 Ammonium sulfate fraction 5,000 67 1.1 AcA34 2,500 55 1.9 GTP-Sepharose 4.6 20 360 -------------------------------------------- ---------- The enzyme activity was measured as follows. PH containing 0.1 mM GTP and 2.5 mM EDTA as reaction solution
5 μl in 495 μl of 8.5 0.1 M Tris-HCl buffer
The mixture was kept at 37 ° C. for 30 minutes, 50 μl of 1N hydrochloric acid containing 0.9% I ₂ and 1.8% KI was added, and the mixture was allowed to stand at room temperature for 1 hour while protecting from light. To this solution was added 50 μl of 2% ascorbic acid, 50 μl of 1N sodium hydroxide and 100 μl of 3 units of alkaline phosphatase solution.
It was kept at 37 ° C. for 1 hour. 100 μl of 1N acetic acid
l was added and the supernatant was obtained after centrifugation. This supernatant was added to 1 mM E
Inject into a 4.6 × 250 mm 5 μm C18 reverse phase column equilibrated with 10 mM sodium phosphate buffer pH 7.0 containing DTA at a flow rate of 0.8 ml / min, excited at 350 nm and using a 440 nm fluorescence detector. Neopterin derived from dihydroneopterin triphosphate, which is a GCH reaction product, is separated and quantified.

The specific activity was measured as follows.
Km 0.05 / μM was determined. That is, GTP is set to 0.0.
1, 0.02, 0.04, 0.06, 0.08, 0.
1, 0.2, 0.4, 0.6, 0.8 and 1.0 μM
The initial rate of GCH was determined using the reaction solution containing
Create a newer-Burk plot and use Km
It was determined.

The molecular weight was measured as follows.
The molecular weight was estimated to be about 26,000 by SDS polyacrylamide electrophoresis analysis of the final chromatographic fraction, and about 200,00 by AcA34 gel filtration.
0, indicating that the enzyme is an oligomer enzyme.

SDS polyacrylamide electrophoresis : L
This was performed using a 12.5% acrylamide gel according to the method of Aemmli. As standard proteins, phosphorylase b (94,000), bovine serum albumin (6
6,000), ovalbumin (43,000), carbonic anhydrase (30,000) and soybean trypsin inhibitor (20,000) were electrophoresed, and the molecular weight of GCH was determined by comparison with the migration position. .

AcA34 gel filtration : Thyroglobulin (669,000), ferritin (440,0) were used as standard proteins under the same conditions using the gel used in the purification process.
00), β-amylase (200,000), alcohol dehydrogenase (150,000), bovine serum albumin (66,000), carbonic anhydrase (2
9,000) and cytochrome c (12,400) were subjected to gel filtration, and the molecular weight of GCH was determined by comparison with the elution position.

The GCH of the final chromatographic fraction was determined for its amino terminal 25 amino acid sequence and internal partial amino acid sequence by the following method.

Determination of amino acid sequence : Purified GCH
540 pmole was precipitated with 10% trichloroacetic acid to give 6
It was dissolved in M guanidine and diluted with a Tris-HCl buffer at pH 9.0 to a final concentration of 50 mM Tris-HCl buffer containing 2 M guanidine. To this, 1/4 of GCH
Then, 00 mol, ie, 1.35 pmole of lysyl endopeptidase was added, and the mixture was kept at 30 ° C. for 5 hours to decompose GCH.
This solution was injected into a 4.6 × 250 mm 5 μm C18 reverse phase column equilibrated with 0.1% trifluoroacetic acid containing 5% acetonitrile, followed by 5% to 60% acetonitrile gradient elution at a flow rate of 1%. 0.0ml
/ Min, gradient 1% / 2ml equilibration buffer at 30 ° C. Detection was performed at 220 nm UV, and the absorption peak fraction was collected in an Eppendorf tube. FIG. 1 shows the results of the elution. The obtained peptide and the amino acid sequence of the undegraded protein were connected to a model 120A high-performance liquid chromatography apparatus using a 470 manufactured by Applied Biosystems.
It was determined using a type A automatic gas phase protein sequencer.

N-terminal of protein Pro-Ser-Leu-Ser-Lys-Glu-Ala-Ala-Leu-Val-His-Glu-Al
The results of amino acid sequencing of the peptide fragment eluted from the a-Leu-Val-Ala-Arg-Gly-Leu-Glu-Thr-Pro-Leu-Arg-Pro 5 μm C18 reverse phase column are as follows. Peak I Ile-Thr-Leu-Ile-Glu-Asn-Lys Peak II Ser-Ser-Gln-Asn-Thr-Arg-His-Gln-Phe-Leu-Arg-Ala-Va
l-Arg-His-His-Asn Peak III Ala- () -Gly-Ile- () -Asp-Ala-Thr-Ser-Ala-Thr Peak IV Glu-Ala-Ala-Leu-Val-His-Glu- Ala-Leu-Val-Ala-Arg-Gl
y-Leu-Glu- Peak V Met-Lys-Val-Asp-Glu-Met-Val-Thr-Val-Arg-Arg-Ile-
() -Leu Peak VI Met-Tyr-Val-Asp-Glu-Ile-Phe-Ser-Gly-Leu-Asp-Tyr-Al
a-Asn-Phe-Pro Peak VII Ser-Leu-Ile-Ala-Gly-His-Met-Thr-Glu-Ile-Met-Gln-Le
u-Leu-Asn- () -Asp Peak VIII Ile-Asn-Arg-Ile-Val-Gln-Phe-Phe-Ala-Gln-Arg-Pro Example 2 Extraction and Purification of PTPS Rat liver 650 g was subjected to male KBL : Obtained from 60 Wister rats. The liver was purified about 32,000-fold by ammonium sulfate fractionation, heat treatment, hydroxyapatite chromatography, butyl toyopearl chromatography, Sephadex G100 gel filtration, and Mono Q ion exchange chromatography as follows. The protein content, activity yield and specific activity in each step are shown in Table 2 below.

Ammonium sulfate fractionation : 650 g of liver stored at -70 ° C. was added with 5 mM 2-mercaptoethanol and 0.2 mM
pH 7 containing mM phenylmethylsulfonylfluorad.
0, 50 mM potassium phosphate buffer (buffer A) 1300
The homogenate was obtained by disruption in a ml using a blender. After this homogenate was mixed with 1300 ml of buffer A, the tissue was further destroyed using a blender manufactured by Ultratrax. This homogenate was added to 1200
After centrifugation at 0 × g for 20 minutes, a supernatant was obtained. This supernatant (2
560 ml) and 1100 ml of 100% saturated ammonium sulfate solution,
After stirring for 20 minutes, the mixture was centrifuged at 12,000 × g for 20 minutes to obtain a supernatant. 1130 ml of 100% saturated ammonium sulfate solution was added to the supernatant, and the mixture was stirred for 20 minutes, centrifuged at 12,000 × g for 20 minutes, and the precipitate was dissolved in a 10 mM potassium phosphate buffer solution (pH 6.0). This enzyme solution was dialyzed by exchanging four times against 5 L of the same buffer.

Heat treatment : 15,000 × dialyzed enzyme solution
The mixture was centrifuged at g for 20 minutes to obtain a supernatant. The sediment has a pH of 1
After dissolving in 0 mM potassium phosphate buffer and centrifuging again under the same conditions, a supernatant was obtained and mixed with the previous supernatant. Add this enzyme solution to 6
Heat-treat while stirring in a hot bath at 80 ° C. in an amount of 0 ml each to obtain 1
Centrifugation was performed at 2000 × g for 20 minutes to obtain a supernatant. The supernatant (347 ml) was concentrated to 79 ml using an ultrafiltration membrane PM-10, and dialyzed against 5 L of 1 mM potassium phosphate buffer (buffer B) at pH 6.0.

[0048]Hydroxyapatite chromatography
ー ： The dialyzed enzyme solution was equilibrated with buffer solution 2.8 ×.
A flow rate of 60 ml /
Adsorbed over time. Transfer this column to 260 ml of buffer B
After washing with water, 700 ml of 1 mM phosphoric acid at pH 6.0 to 200
Perform a gradient elution of mM potassium phosphate buffer
PTPS activity is eluted at about 50 mM phosphate concentration
Was.

Butyl toyopearl chromatography :
The active fraction obtained by hydroxyapatite chromatography was added to a half-volume, 90% saturated ammonium sulfate containing
6.0 of 50 mM potassium phosphate buffer was added. The enzyme solution was adsorbed onto a 1 × 5 cm butyl toyopearl column equilibrated with 50 mM potassium phosphate buffer (buffer C) at pH 6.0 containing 30% saturated ammonium sulfate at a flow rate of 40 ml / hour.
After washing the column with 40 ml of buffer C, a gradient elution from 40 ml of buffer C to buffer C containing no ammonium sulfate eluted PTPS activity with about 20% saturated ammonium sulfate.

Sephadex G100 gel filtration : The active fraction obtained by butyl toyopearl column chromatography was separated by ultrafiltration membrane Amicon Centriflow CF25.
And concentrated to 1.1 ml. The enzyme solution was subjected to gel filtration at a flow rate of 5 ml / hour using a 1.5 × 89 cm Sephadex G100 column equilibrated with 20 mM potassium phosphate buffer (buffer D) containing 30 mM KCl and pH 7.0. An active fraction was obtained.

Mono Q ion exchange chromatography
- : The active fraction obtained by gel filtration was adsorbed onto a 0.5 × 5 cm Mono Q ion exchange column equilibrated with buffer D at a flow rate of 0.5 ml / min. After the column was washed with 3 ml of buffer D, 400 ml of KC was removed from 10 ml of buffer D.
Performing a gradient elution to buffer D containing
PTPS activity eluted as a single peak near the eluate containing 180 mM Cl.

[0052]

[Table 2] Table 2 -------------------------------------------- ------------ Process Protein activity Yield Specific activity (nmol / h (mg) (%) / mg protein) ---------------- ---------------------------------------- Crude extraction 112,000 100 0.68 Ammonium sulfate fraction 41,400 95 1.8 Heat treatment 609 33 41 Hydroxyapatite 47.6 19 300 Butyl toyopearl 4.77 18 2,900 Sephadex G100 0.60 17 22,000 Mono Q 0.57 16 22,000 ----------------------- --------------------------------- The enzyme activity was measured using the product 6-pyruvoyltetrahydro Pterin is iodized in alkali,
The reaction product was quantified as pterin by utilizing the fact that it became pterin.

Measurement of enzyme activity : 50 μM of the reaction solution
Dihydroneopterin triphosphate, 8 mM Mg
PH 7.4 with Cl ₂ and 10 mM dithiothreitol
2 μl of the enzyme solution was added to 18 μl of 100 mM Tris-HCl buffer, incubated at 37 ° C. for 30 minutes, and 200 μl of a 1N sodium hydroxide solution containing 0.9% I ₂ and 1.8% KI was added.
Was added, light was shielded, and the mixture was left at room temperature for 1 hour. 1 in this liquid
200 μl of N hydrochloric acid and 50 μl of 5% ascorbic acid were added, and the supernatant was obtained after centrifugation. This supernatant was washed with 0.1 mM EDTA.
4.6 × 250 mm 5 μm C18 equilibrated with 50 mM sodium acetate buffer, pH 5.0, containing 5% methanol.
Inject into the reverse phase column at a flow rate of 0.8 ml / min.
Pterin derived from 6-pyruvoyltetrahydropterin, which is a PTPS reaction product, was separated and quantified using 0 nm excitation and 440 nm fluorescence detector.

The specific activity was measured as follows.
Km 9.1 μM was determined. That is, dihydroneopterin triphosphate was added to 1, 2, 5, 10, 20 and 50 μl.
Using the above reaction solution containing M, determine the initial velocity of PTPS,
A Lineweaver-Burk plot was created and the Km was determined.

The molecular weight was measured in the same manner as in Example 1 by using SDS.
Performed by polyacrylamide electrophoresis analysis and HPLC gel filtration. However, the gel filtration was carried out using a 50 mM potassium phosphate buffer at pH 7.5 containing 0.1 mM EDTA as an equilibration buffer, using a 1.5 × 47 cm column, and a flow rate of 5 ml / hour. . The molecular weight was estimated to be about 17000 by SDS polyacrylamide electrophoresis analysis of the final chromatographic fraction, and was about 93,000 by HPLC gel filtration. From this result, it is considered that this enzyme is an oligomer enzyme.

The amino acid sequence of the amino terminal 24 and the partial amino acid sequence inside the PTPS of the final chromatographic fraction were determined by the following method.

Determination of amino acid sequence : purified PTPS 1
Using Nmol, the N-terminal amino acid sequence was determined in the same manner as in the determination of the amino acid sequence in Example 1. Purified PTPS
(1 nmol), peptide separation and amino acid sequence determination were also performed in the same manner as in Example 1. However, the enzymatic degradation was carried out by V8 protease in addition to lysyl endopeptidase, and the latter degradation reaction was carried out by PTPS.
After precipitation of 1 nmol of trichloroacetic acid, the precipitate was dissolved in 6 μl of 6 M guanidine, and then 10 μl of 0.5 M ammonium acetate buffer (pH 7.7) and 8.2 μl of H ₂ O were added.
8 protease was added at 5 pmole and reacted at 37 ° C. for 12 hours. This degradation product was decomposed into peptides in the same manner as in Example 1, and the amino acid sequence was determined. FIGS. 2 and 3 show the results of eluting a peptide degraded with lysyl endopeptidase and V8 protease, respectively, on a C18 reverse phase column in the same manner as in Example 1. Protein N-terminal _{NH 2 -Val-Gly-Leu-} Arg-Arg-Arg-Ala-Arg-Leu-Ser-Arg-Le
The results of amino acid sequencing of the peptide fragment eluted from the u-Val-Ser-Phe- () -Ala- () -His-Arg-Leu-His- () -Pro C18 reverse phase column are as follows.

Lys I Pro-Leu-Asn-His- () -Lys Lys II () -Asn-Asn-Pro-Asn-Gly-His-Gly- () -Asn-As
p Lys III Val-Phe-Gly-Lys Lys IV Val-Tyr-Glu-Thr-Asp-Asn-Asn-Ile-Val-Val-Ty
r-Lys-Gly-Glu Lys V Glu-Tyr-Met-Glu-Glu-Ala-Ile-Met-Lys-Pro-Le
u-Asp Lys VI Val-Val-Val-Thr-Ile-His-Gly-Glu-Ile-Asp-Pr
o-Val-Thr-Gly-Met-Val- () -Asn-Leu V8 I Thr-Asp-Asn-Asn-Ile-Val-Val-Tyr-Lys-Gly-Gl
u V8 II Asn-Leu-Gln-Arg-Leu-Leu-Pro-Val-Gly-Ala-Le
u-Tyr V8 III Ala-Ile-Met-Lys-Pro-Leu-Asp-His-Lys-Asn-Le
u-Asp- ()-() -Val V8 IV Ile-Asp-Pro-Val-Thr-Gly In the above sequences, parentheses are unidentified amino acids. Example 3 Extraction and Purification of SPR Rat erythrocytes were converted to male KBL: Wister rat 3
Obtained from 0 animals. The obtained erythrocytes are subjected to hemolysis, ammonium sulfate fractionation, butyl toyopearl chromatography, hydroxyapatite chromatography, Red Sepharose CL-6
Purification was performed about 1100 times by B gel filtration as follows.
The protein content, activity yield and specific activity of each step are shown in Table 3 below.

Hemolysis : Approximately 116 g of rat erythrocytes stored at -70 ° C. were combined with 466 ml of 5 mM potassium phosphate buffer (buffer A) at pH 7.0 containing 0.2 mM phenylmethylsulfonyl fluoride (buffer A). Mix in the blender,
After stirring on ice for 1 hour, the mixture was centrifuged at 9,500 × g for 30 minutes to obtain an extract (1). The precipitate was again stirred in 291 ml of buffer A for 1 hour and then centrifuged in the same manner to obtain an extract (2). Extracts (1) and (2) were mixed.

Ammonium sulfate fractionation : Extract 710 obtained by hemolysis
163 g of ammonium sulfate was added to the mixture, and the mixture was stirred for 20 minutes.
After centrifugation at 000 × g for 30 minutes, 69.5 g of ammonium sulfate was added to 780 ml of the supernatant, and the mixture was stirred for 20 minutes, and then 13,000 ×
The mixture was centrifuged at g for 30 minutes to obtain a precipitate. 53 ml of this sediment
It was dissolved in 10 mM potassium phosphate buffer at pH 6.0.

Butyl toyopearl chromatography :
5. pH 53 containing 53 ml of ammonium sulfate fraction and 53 ml of 60% saturated ammonium sulfate.
0 of 50 mM potassium phosphate buffer. This enzyme solution was adsorbed at a flow rate of 40 ml / h onto a 10 ml butyl toyopearl column equilibrated with a 50 mM potassium phosphate buffer at pH 6.0 containing 30% saturated ammonium sulfate. 10 ml in this column
Buffer containing 30% saturated ammonium sulfate and 10 ml of 15%
After flowing the same buffer containing saturated ammonium sulfate in order, the enzyme was
When eluted with a gradient of 100 ml of the same buffer from saturated ammonium sulfate to 0% ammonium sulfate, sepiapterin reductase activity was eluted at about 8% saturated ammonium sulfate.

[0062]Hydroxyapatite chromatography
ー: 12 ml of the active fraction eluted with butyltoyopearl
Using 200 ml of ADEX G-25, pH 6.0
After desalting in 0 mM potassium phosphate buffer, equilibrate with the same buffer
Adsorbed on a 1.0 ml hydroxyapatite column
I let you. Wash the unadsorbed fraction with 10 ml of the same buffer.
After purification, potassium phosphate buffer from 10 mM to 200 mM
A 50 ml gradient elution results in a phosphoric acid concentration of about 80
Sepiapterin reductase activity was eluted at mM
Was.

Red Sepharose CL-6B gel filtration :
1. To 2.8 ml of the active fraction eluted with hydroxyapatite
8 ml H ₂ O was added and adsorbed on a 1 ml Red Sepharose column equilibrated with 20 mM potassium phosphate buffer at pH 6.8. The column was washed with 2 ml of the same buffer to remove unadsorbed components, and then 10 ml of a 50 mM potassium phosphate buffer (pH 6.8) containing 0.1 M KCl was passed, followed by 50 mM N 2
Sepiapterin reductase activity was eluted with the same buffer containing ADPH.

[0064]

[Table 3] Table 3 -------------------------------------------- ----------------- Step Protein mass Activity yield Specific activity (nmol / h (mg) (%) / mg protein) ----------- -------------------------------------------------- Hemolysis 2500 100 460 Ammonium sulfate fraction 160 94 6,600 Butyl toyopearl 8.8 48 66,000 Hydroxyapatite 0.53 13 270,000 Red Sepharose CL-6B 0.29 13 520,000 --------------------- ---------------------------------------- The enzyme activity is measured as follows. went. PH 6.4 containing 50 μM sepiapterin, 100 μM NADPH and 0.1 mg / ml bovine serum albumin as a reaction solution.
2.5 to 47.5 μl of 100 mM potassium phosphate buffer
After adding 10 μl of the enzyme solution and incubating at 25 ° C. for 10 minutes,
μl of an acidic iodine solution was added, and the mixture was allowed to stand at room temperature for 1 hour while protected from light. Subsequently, 250 μl of 1% ascorbic acid was added, followed by high performance liquid chromatography using a 5 μm C18 column (equilibration buffer was pH 5 containing 5% methanol).
Use 50 mM sodium acetate buffer at 5.0, flow rate 0.
(8 ml / min, analyzed at 25 ° C.) to separate and quantitate biopterin, which is an oxide of the enzyme reaction product dihydrobiopterin.

In addition, Km 9.4 μM with respect to sepiapterin was determined as follows. That is, sepiapterin 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 75 and
Using the above reaction solution containing 100 μM, the initial rate of sepiapterin reductase was determined, and Lineweaver-Burn was determined.
A plot of k was made and Km was determined.

The molecular weight was measured by the same SDS polyacrylamide electrophoresis as in Example 1. The molecular weight was estimated to be approximately 28,800 by SDS polyacrylamide electrophoresis analysis of the SPR obtained by final gel filtration. Also H
According to PLC gel filtration, the molecular weight was about 56,000. From these results, this enzyme is considered to be a dimer enzyme.

The partial amino acid sequence of SPR in the final chromatographic fraction was determined in the same manner as in Example 1 using 2 nmole of the purified SPR sample. FIG. 4 shows the results of eluting SPR degraded with lysyl endopeptidase using a C18 reverse phase column in the same manner as in Example 1. The results of amino acid sequencing of the peptide separated and eluted from the column were as follows.

Peak I Leu-Leu-Ser-Leu-Leu-Gln-Arg-Asp-Thr-Phe-Gln-Ser-Gl
y-Ala-His-Val-Asp-Phe-Tyr-Asp-Ile Example 4 Measurement of Stable and Optimal pH The relationship between pH and enzyme activity was examined as follows. (1) For GCH, using the reaction solution composition shown in Example 1, replacing only the buffer with 50 mM sodium acetate, MES, potassium phosphate, Hepes, Tris or sodium glycine buffer, and using 0.1 M KCl Examined in the presence or absence. (2) As for (1), PTPS was performed under the reaction conditions shown in Example 2 except that only the buffer solution was changed. No effect of 0.1 M KCl was observed on this enzyme. (3) As for SPR, at 75 mM, only the buffer solution was changed as in (1) and 0.1 M
Examination was performed in the presence or absence of KCl.

FIG. 5 shows the results. As is clear from FIG. 5, it can be seen that GCH, PTPS and SPR have an optimum pH around 7. The meaning of the plot in FIG. 5 is as shown in the following table.

[0070]

[Table 4]

The buffer concentrations were tested at 50 mM for GCH and PTPS and 75 mM for SPR. Example 5 Production of BH4 by One Pot Method BH4 was synthesized by the following composition.

A 50 mM potassium phosphate buffer (pH 7.
0) 0.2 mM GTP 0.1 M potassium chloride 10 mM dithioerythritol 4 mM NADPH 8 mM MgCl ₂ 1 mM EDTA 770 μg / ml ^* Escherichia coli-derived GCH cyclohydrolase I 96 μg / ml ^* Rat liver-derived 6-pyruvoyltetrahydropterin synthesis Enzyme 8 μg / ml ^* Rat erythrocyte-derived sepiapterin reductase ( ^* final concentration in reaction solution) Prepare a reaction solution of the above composition, add a mixture of the three enzymes at the end, and react at 37 ° C for 20 minutes. 0.2N trichloroacetic acid containing 5 mM dithiothreitol (DTT) was added, and the mixture was centrifuged to obtain a supernatant. The reaction product is a strong cation ion exchange resin (Whatman SC
X) A 4.6 × 250 mm column was run with a pH of 5% methanol.
Equilibrate with 3.8 0.1 M ammonium acetate buffer,
Analysis was performed at a flow rate of 1 ml / min. Detection was performed at a voltage of 300 mV using an electrochemical detector. FIG. 6 shows the result. By adding 1 mM DTT to this separation system, BH
4 was separated and recovered stably.

FIG. 7 shows the results of the above reaction over time.
The yield of BH4 from starting GTP was about 75%. Example 6 Analysis of Partial Sequence of Rat GTP Cyclohydrolase I Hatakeyama, K., Hara
da, T .; Suzuki, S .; , Watanabe,
Y. , And Kagamiyama, H .; (1989)
J. Biol. Chem. 264 , 21660-216
64), rat GTP cyclohydrolase I was purified and digested with lysyl endopeptidase (GTP
Cyclohydrolase I / lysyl endopeptidase = 4
00/1 (molar ratio), 30 ° C, 7 hours). The decomposition products are
Using a 5 μm C18 column (manufactured by Nacalai Tesque) equilibrated with 0.1% trifluoroacetic acid-5% acetonitrile, a linear concentration gradient of 5-40% acetonitrile (25
At a flow rate of 1.0 ml / min for 60 minutes) at 60 ° C. (FIG. 8). The sequence of the fractionated peptide was analyzed using an automatic gas phase protein sequence analyzer (Applied Biosystems, Model 470A).

Synthesis of oligodeoxyribonucleotides Oligodeoxyribonucleotides were produced by an automatic phosphoramidite method using a DNA synthesizer (Model 381A, manufactured by Applied Biosystems). These oligonucleotides were labeled with T4 polynucleotide kinase and [γ- ³² P] ATP.

Synthesis of cDNA and cDNA Library
Screening total RNA was extracted from rat liver (Chirgwin,
J. M. , PrzyByla, A .; E. FIG. , MacDon
ald, R .; J. And Rutter, W.C. J. (1
979) Biochemistry 18 , 5294.
5299). Poly (A) RNA was isolated by subjecting the extracted total RNA to oligo (dT) -cellulose chromatography (Aviv, H., and Leder, P. et al.
(1972) Proc. Natl. Acad. Sci.
ScL USA, 69 , 1408-1412). Double-stranded cDNA
Was synthesized using a cDNA synthesis kit (Pharmacia LKB Biotechnology) according to the method of Gubler and Hoffman (Gubler, V., and Hoffman, BJ. (1983) Gene.
(Amst.) 25 , 263-269). The blunt-ended double-stranded cDNA was generated according to the method of Crickstein et al. (Klickstein, LB and N) except that RNase T1 (0.1 μg / mg) was additionally used.
eve, R.E. L (1987) in Current P
rotocols in Molecular Bio
logic (Ausubel, FM, Brent,
R. Kingston, R .; E. FIG. Moore, D .;
D. , Seidman, J .; G. FIG. , Smith, J .;
A. And Struhl, K .; , Eds), pp
5.5.-5.5.10, Greene Publi
shing Associates and Wiley
-Interscience, New York). c
After the EcoRI / NotI adapter (cDNA synthesis kit: manufactured by Pharmacia) was ligated to each blunt end of the DNA, the cDNA was inserted into the EcoRI site of λZAPII vector DNA (manufactured by Stratagene). The obtained library DNA was packaged in vitro using Gigapack Gold (manufactured by Stratagene), and introduced into Escherichia coli XL1-Blue.

Replica Nylon Filter (Dupont-
GeneSc from New England Nuclear
About 3.0 × 10 ⁵ plaques were screened with ³² P-labeled synthetic oligonucleotide probes (Benton, WD, and Davis, RW (1977) Science 1 ) using Reen Plus).
96 , 180-182). The base sequences of the three probes are deduced from the amino acid sequences of the lysyl endopeptidase-treated peptides of GTP cyclohydrolase I, as described later. One type of probe is codon usage analysis (Lathe, R. (1985) J. Mo.
l. Biol. 183 , 1-12) and two other types of probes, a mixture of short oligonucleotides having all codon combinations, were used as other types of probes. The replica nylon filter is SS containing 5 volumes of 0.1% sodium dodecyl sulfate (SDS).
C (containing SSC solution: 0.15 M sodium chloride, 15 mM sodium citrate), 10 volumes of Denhardt
t solution (Denhardt solution: containing 0.2% (W / V) ficoll, polyvinylpyrrolidone, fetal bovine serum albumin) and 0.1 mg / ml calf thymus DNA
(Sigma) at 65 ° C. for at least 3 hours. A radiolabeled oligonucleotide probe was added to the solution and the filters were hybridized at 42 ° C. overnight (Maniatis,
T. Fritsch, E .; F. , And Sambro
ok, J. et al. (1982) Molecular Clon
ing: A Laboratory Manual, G
old Spring Harbor Laborat
ory, Cold SpringHarbor, N
Y). The filters were washed at 40 ° C. with 0.2 volumes of SSC containing 0.1% SDS to remove long oligonucleotides, while 4 volumes of 0.1% SDS containing 0.1% SDS to remove short oligonucleotides. After washing with SSC,
It was subjected to autoradiography. Hybridization-Positive clones were plaque purified and phage were purified from the Stratagene automatic execution protocol.
xcision protocol) and Blue
Converted to script plasmid.

Analysis of Nucleotide Sequence The cDNA insert was subcloned into the EcoRI site of the M13 vector (Vieira, J. and Messing, J. (1987) Meth. Enzy.
mol. 153 , 3-11). The nucleotide sequence of the insert was determined using Sequence kit Ver. 2.0
(7-denza-dGTP-edition, Uni
determined by the dideoxy chain termination method using ted States Biochemical and synthetic primers (Sanger, F., Ni
cklen, S.M. And Coulson, A .; R.
(1977) Proc. Natl. Acad. Sci.
USA 74 , 5463-5467).

Sequence Analysis by Computer The amino acid sequence deduced from the cDNA sequence was obtained from a database system (GENA) developed by Kuhara et al.
S) (Kuhara, S., Matsuuo, F., Fu
tamura, S.M. , Fugita, A .; , Shino
hara, T .; Takagi, T .; , And Saka
ki, Y. (1984) Nucleic Acids R
es. 12 , 89-99), by the programs of Wilbur and Lippman (Wilbur, WJ, and Lip).
man, D .; J. (1983) Proc. Natl. A
cad. Sci. USA, 80 , 726-730).

(Result) Partial amino acid sequence of rat GTP cyclohydrolase I Eight peptides were isolated (I to VIII in FIG. 8), and their complete or partial amino acid sequence was determined (FIG. 10).
Is underlined). The N-terminal sequence of the purified enzyme was determined up to 30 residues: Gly-Phe-Pro-Glu-Arg-Glu-Leu-Pro-Arg-Pro-Gly-Ala-Se
r-Arg-Pro-Ala-Glu-Lys-Ser- () -Pro-Pro-Gln-Ala-Lys-
Gly-Ala-Gln-Pro-Ala.

The twentieth amino acid in the above sequence was determined to be Arg from the cDNA (see FIG. 10).

From the amino acid sequence of the isolated proteinaceous peptide of the cDNA clone, three single long-chain oligonucleotides having a unique sequence [5′-TTCC
AGGCATCAGCAGGCTGGGCACCTTTGGCTTCAGG (from peptide I and part of the protein N-terminal sequence in FIG. 10: probe 1);
5'-TTGGTGAAGAACTGCATGGCTGTGGCAGCTCTCCATGGGGT (peptide IV: probe 2); 5'-ACGCCAGCAGGCTGCAGGGCCT
CTGTGATGGCCACAGCAATCTG (peptide VIII: probe 3)] and a mixture of two short oligonucleotides covering all possible codon combinations [5'-
TTCCA (G / A / T / C) GC (G / A) TC (G / A / T / C) GC (Peptide I:
Probe 4); 5'-GT (G / A) AA (G / A) AA (T / C) TGCAT (G / A / T
/ C) GC (peptide IV: probe 5)]. These clones were isolated from rat liver poly (A) ⁺ RNA-derived λZ
It was used for screening about 3 × 10 ⁵ clones of the APII cDNA library. Four clones were hybridized with probes 2, 3 and 5.
Probe 4, one of the short-chain probes, was not detected because the signal of λZAP plaque was very weak, and it is considered that it did not hybridize with these clones. In addition, in probe 1, which is one of the long-chain oligonucleotide probes, it is considered that 8 bases (21%) of the present probe were erroneously estimated and did not hybridize with these clones. These clones were partially sequenced and found to be identical except for the length of the 5'- and 3'-untranslated regions. The longest of these clones contained a poly (dA) tract, so this clone was subjected to further analysis.

Complete Nucleotide Sequence The primary sequence of the rat GTP cyclohydrolase I cDNA was determined for both chains according to the strategy shown in FIG. The result is shown in FIG. The inserted fragment is poly (A)
⁺ 1024 nucleotides, including the 8 adenine residues of the tail. Open reading frame is 723
And the subsequent 3'-untranslated region was 163 nucleotides from the stop codon TGA to the poly (A) tail. There was a polyadenylation signal AATAAA 13 nucleotides upstream from the poly (A) tail of the 3'-untranslated region. The open reading frame encoded a polypeptide of 241 amino acids, starting from the first ATG in the sequence of the clone. Sequences around the start codon are sequences that are preferred as eukaryotic start sites (Kozak, M .;
(1984) Nucleic. Acids Res. 1
2 , 857-872). The amino acid sequence deduced from the nucleotide sequence is shown in FIG. The amino acid sequences of the eight proteinaceous peptides matched the sequences deduced from the nucleotide sequences. N of the produced protein shown above
The terminus completely coincided with residues 12 to 41 of the deduced amino acid sequence. Furthermore, the amino acid composition of the purified enzyme was in good agreement with the composition of the sequence deduced from residues 12 to 241 (Table 5).

[0083]

TABLE 5 Amino acid composition of purified rat GTP cyclohydrolase I and amino acid composition deduced from primary sequence of cDNA ---------------------- Number of residues Amino acids ------------------------ Analytical value ^a) Estimated value ------------------------------------------ Arginine 18.21 18 lysine 11.85 12 histidine 5.25 5 aspartic acid 16.58 11 asparagine 6 threonine 11.74 12 serine 17.00 13 glutamic acid 29.82 19 glutamine 11 proline 14.33 15 glycine 18.02 14 alanine 20.98 20 cysteine- ^b) 2 valine 16.00 17 methionine 7.33 9 isoleucine 11.12 12 leu Tyrosine 4.11 4 Phenylalanine 7.96 8 Tryptophan- ^b) 2 --------------------------------------- --- ^a) amino acid analysis 110 ° C. the protein, after acid hydrolysis of 24 hours with a Hitachi L-8500 Model amino acid analyzer, O- Futaruaru It was carried out by post-column label method of hydrate.

^B) Not measured. Therefore, rat GTP cyclohydrolase I contains 2
It has 30 amino acid residues and has a calculated molecular weight of 25,
815; this value is one unit from the molecular weight of the purified protein reported by Hatakeyama et al. (Supra).
4% smaller. This may be because post-translational cleavage at the Asn ₁₁ -Gly ₁₂ bond converts the putative primary polypeptide to the mature form of the enzyme. This presequence has three basic and one acidic residue, is net positively charged, but lacks the long hydrophobic region common to signal peptides of secreted proteins. Therefore, the function of this front sequence cannot be estimated. The other three hybridization-positive clones gave similar results.

The estimated protein sequence was
Chemical Research Foundation (Nati
onal Biochemical Research
 Foundation) protein sequence database and
By comparison, 77-96 amino acid residues were dihydro
Residues in the high conversion region of borate reductase (Beverle
y, S. M. , Ellenberger, T .; E. FIG. ， O
And Cordingley, J .; S. (1986) Pr
oc. Natl. Acad. Sci. U. S. A.8
3Grummont, R .; , Wa
shtien, W.C. L. , Caput, D.A. , And S
anti, D .; V. (1986) Proc. Natl.
Acad. Sci. U. S. A.83, 5387-53
91) and a significant homology (Fig.
11). These residues are used by dihydrofolate reductase
Binding of methotrexate or dihydrofolate to
Borin et al. And Matthews et al.
It has been crystallographically identified (Bolin, JT,
Filman, D.M. J. Matthews, D .;
A. Hamlin, R .; C. And Kraut, J. et al.
(1982) J. Am. Biol. Chem.257, 136
50-13662; Matthews, D .; A. , B
olin, J .; T. Burridge, J .; M. , F
ilman, D .; J. Volz, K .; W. , Kauf
man, B .; T. , Bedell, C .; R. , Cham
pness, J.M. N. , Stammers, D.A. K. ,
And Kraut, J. et al. (1985) J. Amer. Biol. C
hem.260381-391). In particular, FIG.
Tryptophan and phenylalanine
Interacts with the pterin ring of these compounds
(See Borin et al. And Matthews et al., Supra). Correspondence
Liptophan and phenylalanine residues are
It is found in the sequence of crohydrolase I. GTP cyclo
The activity of hydrolase I depends on the activity of dihydrofolate reductase.
Compared to the Km value for dihydrofolate,
With a Ki value of 12 μM and 170 μM
Such as hydrobiopterin and dihydrofolate
Inhibited by thelins (Shen, R., Ala
m, A. , And Zhang, Y .; (1988) Bio
chim. Biophys. Acta965, 9-1
5) The dihydrofolate reductase is 7,8-dihydro
Reduction of biopterin to tetrahydropterin (Ka
ufman, S .; (1967) J. Mol. Biol. Che
m.242, 3934-3943). Biopterin and leaves
Since these acid salts have a common pterin moiety, these results
Means that GTP cyclohydrolase I
Pterin binding site similar to the pterin binding site of the original enzyme
And 77-9 of the deduced amino acid sequence
Residue consisting of residue 6 can respond to pterin binding
Suggests a site.

If site-specific mutation is performed at the pterin-binding site deduced from the results, rat GTP cyclohydrolase I which is not inhibited by BH4 may be produced by genetic engineering.

[0087]

Industrial Applicability According to the present invention, 6- (R) -L useful for treating a disease caused by a monoamine neurotransmitter deficiency disorder.
-Erythro-5,6,7,8, -tetrahydrobiopterin (BH4) can be efficiently produced by a simple operation.

The present invention can also be advantageously used for producing ¹⁴ C-labeled BH4 as a probe useful for understanding the physiological function of BH4.

Further, the present invention discloses an enzyme having a new structure (amino acid sequence), thereby isolating a gene of the enzyme using a DNA encoding the enzyme as a probe.
Furthermore, BH4, which cannot be produced by microorganisms in a cell or a material for mass production of the enzyme of the present invention by recombinant DNA technology.
And DNA for synthesizing the same.

[0090]

[Sequence List] SEQUENCE LISTING <110> SUNTORY LIMITED <120> Enzymes for producing tetrahydrobiopterin <130> 000314 <160> 31 <210> SEQ ID NO: 1 <211> 25 <212> PRT <213> E. coli <220 > <223> N-terminal region of E. coli GTP cyclohydrolase I <400> 1 Pro Ser Leu Ser Lys Glu Ala Ala Leu Val His Glu Ala Leu Val 1 5 10 15 Ala Arg Gly Leu Glu Thr Pro Leu Arg Pro 20 25 <210> SEQ ID NO: 2 <211> 15 <212> PRT <213> rat <220> <223> N-terminal region of rat liver 6-pyruvoyltetrahydropterin synthetase <400> 2 Val Gly Leu Arg Arg Arg Ala Arg Leu Ser Arg Leu Val Ser Phe 1 5 10 15 <210> SEQ ID NO: 3 <211> 21 <212> PRT <213> rat <220> <223> partial sequence of rat erythrocyte sepiapterin reductase <400> 3 Leu Leu Ser Leu Leu Gln Arg Asp Thr Phe Gln Ser Gly Ala His 1 5 10 15 Val Asp Phe Tyr Asp Ile 20 <210> SEQ ID NO: 4 <211> 7 <212> PRT <213> E. coli <220> <223 > peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 4 Ile Thr Leu Ile Glu Asn Lys 1 5 <210> SEQ ID NO: 5 <211> 1 7 <212> PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 5 Ser Ser Gln Asn Thr Arg His Gln Phe Leu Arg Ala Val Arg His 1 5 10 15 His Asn <210> SEQ ID NO: 6 <211> 11 <212> PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 6 Ala Xaa Gly Ile Xaa Asp Ala Thr Ser Ala Thr 1 5 10 <210> SEQ ID NO: 7 <211> 15 <212> PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 7 Glu Ala Ala Leu Val His Glu Ala Leu Val Ala Arg Gly Leu Glu 1 5 10 15 <210> SEQ ID NO: 8 <211> 14 <212> PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 8 Met Lys Val Asp Glu Met Val Thr Val Arg Arg Ile Xaa Leu 1 5 10 <210> SEQ ID NO: 9 <211> 16 <212 > PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 9 Met Tyr Val Asp Glu Ile Phe Ser Gly Leu Asp Tyr Ala Asn Phe Pro 1 5 10 15 <210> SEQ ID NO: 10 <211> 17 <212> PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 10 Ser Leu Ile Ala Gly His Met Thr Glu Ile Met Gln Leu Leu Asn 1 5 10 15 Xaa Asp <210> SEQ ID NO: 11 <211> 12 <212 > PRT <213> E. coli <220> <223> peptide fragment of E. coli GTP cyclohydrolase I digested with lysylendopeptidase <400> 11 Ile Asn Arg Ile Val Gln Phe Phe Ala Gln Arg Pro 1 5 10 <210> SEQ ID NO: 12 <211> 24 <212> PRT <213> rat <220> <223> N-terminal region of rat liver 6-pyruvoyltetrahydropterin synthetase <400> 12 Val Gly Leu Arg Arg Arg Ala Arg Leu Ser Arg Leu Val Ser Phe 1 5 10 15 Xaa Ala Xaa His Arg Leu His Xaa Pro 20 <210> SEQ ID NO: 13 <211> 6 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 13 Pro Leu Asn His Xaa Lys 1 5 <210> SEQ ID NO: 14 <211> 11 <212> PR T <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 14 Xaa Asn Asn Pro Asn Gly His Gly Xaa Asn Asp 1 5 10 <210> SEQ ID NO: 15 < 211> 4 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 15 Val Phe Gly Lys 1 <210> SEQ ID NO: 16 <211> 14 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 16 Val Tyr Glu Thr Asp Asn Asn Ile Val Val Tyr Lys Gly Glu 1 5 10 <210> SEQ ID NO: 17 <211> 12 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 17 Glu Tyr Met Glu Glu Ala Ile Met Lys Pro Leu Asp 1 5 10 <210> SEQ ID NO: 18 <211> 19 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with lysylendopeptidase <400> 18 Val Val Val Thr Ile His Gly Glu Ile Asp Pro Val Thr Gly Met 1 5 10 15 Val Xaa Asn Leu <210> SEQ ID NO: 19 <211> 11 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with V8 protease <400> 19 Thr Asp Asn Asn Ile Val Val Tyr Lys Gly Glu 1 5 10 <210> SEQ ID NO: 20 <211> 12 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with V8 protease <400> 20 Asn Leu Gln Arg Leu Leu Pro Val Gly Ala Leu Tyr 1 5 10 <210> SEQ ID NO: 21 <211> 15 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with V8 protease <400> 21 Ala Ile Met Lys Pro Leu Asp His Lys Asn Leu Asp Xaa Xaa Val 1 5 10 15 <210> SEQ ID NO: 22 <211> 6 <212> PRT <213> rat <220> <223> peptide fragment of rat liver 6-pyruvoyltetrahydropterin synthetase digested with V8 protease <400> 22 Ile Asp Pro Val Thr Gly 1 5 <210> SEQ ID NO: 23 <211> 21 <212> PRT <213> rat <220> <223> peptide fragment of rat erythrocyte sepiapterin reductase digested with lysylendopeptidase <400> 23 Leu Leu Ser Leu Leu Gln Arg Asp Thr Phe Gln Ser Gly Ala His 1 5 10 15 Val Asp Phe Tyr Asp Ile 20 <210> SEQ ID NO: 24 <211 > 30 <212> PRT <213> rat <220> <223> N-terminal region of rat GTP cyclohydrolase I <400> 24 Gly Phe Pro Glu Arg Glu Leu Pro Arg Pro Gly Ala Ser Arg Pro 1 5 10 15 Ala Glu Lys Ser Xaa Pro Pro Gln Ala Lys Gly Ala Gln Pro Ala 20 25 30 <210> SEQ ID NO: 25 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> probe for cloning rat GTP cyclohydrolase I <400> 25 TTCCAGGCAT CAGCAGGCTG GGCACCTTTG GCTTCAGG 38 <210> SEQ ID NO: 26 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> probe for cloning rat GTP cyclohydrolase I <400> 26 TTGGTGAAGA ACTGCATGGC TGTGGCAGCT CTCCATGGGG T 41 <210> SEQ ID NO: 27 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> probe for cloning rat GTP cyclohydrolase I <400> 27 ACGCCAGCAG GCTGCAGGGC CTCTGTGATG GCCACAGCAA TCTG 44 <210> SEQ ID NO: 2 8 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> probe for cloning rat GTP cyclohydrolase I <400> 28 TTCCANGCRT CNGC 14 <210> SEQ ID NO: 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> probe for cloning rat GTP cyclohydrolase I <400> 29 GTRAARAAYT GCATNGC 17 <210> SEQ ID NO: 30 <211> 897 <212> DNA <213> rat <220> <223 > cDNA sequence of rat GTP cyclohydrolase I <400> 30 GACTTCGAAC CTCATTCGGT GCAGAACTCC TGTCCCGGTG ACAGCCACAG GTCACGGCCG -68 CCGGCTAAGC CGAGCCGCAG CGCTTGGTAG CACCTTAGGG TGTCTCGGGA GCAATCGCGC -8 CGGGTCC ATG GAG GAG GAG GAG GAG AGGTCAG GAG AGT GCC AGG GAG GAG GAG GAG AGC GGG CAG GAG GAG GAG GAG GAG GAG AGC GAG GAG AGT GCC AGG CGG Val Arg Cys Thr Asn Gly Phe Pro 1 5 10 GAG CGG GAG CTG CCG CGG CCC GGG GCC AGC CGA CCT GCC GAG AAG TCC 90 Glu Arg Glu Leu Pro Arg Pro Gly Ala Ser Arg Pro Ala Glu Lys Ser 15 20 25 30 CGG CCG CCC GAG GCC AAG GGC GCA CAG CCA GCC GAC GCC TGG AAG GCA 138 Arg Pro Pro Glu Ala Lys Gly Ala Gln Pro Ala Asp Ala Trp Lys Ala 35 40 45 GGG CGG CCC CGC AGC GAG GAG GAT AAC GAG CTG AAC CTC CC C AAC CTG 186 Gly Arg Pro Arg Ser Glu Glu Asp Asn Glu Leu Asn Leu Pro Asn Leu 50 55 60 GCG GCC GCT TAC TCG TCC ATC CTG CGC TCG CTG GGC GAG GAC CCC CAG 234 Ala Ala Ala Ala Tyr Ser Ser Ile Leu Arg Ser Leu Gly Glu Asp Pro Gln 65 70 75 CGG CAG GGG CTG CTC AAG ACG CCC TGG AGG GCG GCC ACC GCC ATG CAG 282 Arg Gln Gly Leu Leu Lys Thr Pro Trp Arg Ala Ala Thr Ala Met Gln 80 85 90 TTC TTC ACC AAG GGA TAC CAG GAG ACC ATC TCA GAT GTC CTG AAC GAT 330 Phe Phe Thr Lys Gly Tyr Gln Glu Thr Ile Ser Asp Val Leu Asn Asp 95 100 105 110 GCT ATA TTT GAT GAG GAC CAT GAC GAG ATG GTG ATT GTG AAG GAC ATT 378 Ala Ile Phe Asp Glu Asp His Asp Glu Met Val Ile Val Lys Asp Ile 115 120 125 GAC ATG TTT TCC ATG TGT GAG CAT CAC CTG GTC CCA TTT GTG GGA AGG 426 Asp Met Phe Ser Met Cys Glu His His Leu Val Pro Phe Val Gly Arg 130 135 140 GTC CAT ATT GGT TAT CTT CCT AAC AAG CAA GTC CTT GGT CTC AGC AAA 474 Val His Ile Gly Tyr Leu Pro Asn Lys Gln Val Leu Gly Leu Ser Lys 145 150 155 CTT GCC AGG ATT GTG GAA ATC TAC AGT AGA AGA CTA CAA GTT CAA GAA 522 Leu Ala Arg Ile Val Glu Ile Tyr Ser Arg Arg Leu Gln Val Gln Glu 160 165 170 CGC CTT ACC AAA CAG ATT GCA GTG GCC ATC ACA GAA GCC TTG CAG CCT 570 Arg Leu Thr Lys Gln Ile Ala Val Ala Ile Thr Glu Ala Leu Gln Pro 175 180 185 190 GCT GGC GTC GGG GTA GTG ATT GAA GCA ACA CAC ATG TGT ATG GTC ATG 618 Ala Gly Val Gly Val Val Ile Glu Ala Thr His Met Cys Met Val Met 195 200 205 CGA GGT GTG CAG AAA ATG AAC AGC AAG ACT GTC ACT AGC ACC ATG CTA 666 Arg Gly Val Gln Lys Met Asn Ser Lys Thr Val Thr Ser Thr Met Leu 210 215 220 GGC GTG TTC CGG GAA GAC CCA AAG ACT CGG GAG GAG TTC CTC ACA CTC 714 Gly Val Phe Arg Glu Asp Pro Lys Thr Arg Glu Glu Phe Leu Thr Leu 225 230 235 ATC AGG AGC TGA ACTTCCGTGT GCGAGCCCCG GTTTGCAGAC CCCCGCTGAG 766 Ile Arg Ser 240 GCCAGCGTTA TCTGTCA TGATCTA TGATCTA TGATCTAGA TTGTACATTA 8 > SEQ ID NO: 31 <211> 20 <212> PRT <213> rat <220> <223> partial sequence of rat GTP cy clohydrolase I <400> 31 Pro Gln Arg Gln Gly Leu Leu Lys Thr Pro Trp Arg Ala Ala Thr 1 5 10 15 Ala Met Gln Phe Phe 20

[Brief description of the drawings]

FIG. 1 is a diagram showing a state in which GCH was decomposed with lysyl endopeptidase in Example 1 and then eluted with acetonitrile by C18 reverse phase column chromatography.

FIG. 2 is a diagram showing a state in which PTPS was decomposed with lysyl endopeptidase in Example 2 and then eluted with acetonitrile by C18 reverse phase column chromatography.

FIG. 3 is a diagram showing a state in which PTPS was degraded with V8 protease in Example 2 and then eluted with acetonitrile by C18 reverse phase column chromatography.

FIG. 4 is a diagram showing a state in which SPR was decomposed with lysyl endopeptidase in Example 3 and then eluted with acetonitrile by C18 reverse phase column chromatography.

FIG. 5 is a graph showing the optimum pH of GCH, PTPS and SPR.

FIG. 6 is a view showing a state in which BH4 synthesized by a one-pot method in Example 5 is isolated by a Whatman SCX column.

FIG. 7 is a one-pot method of Example 5 for BH.
4 is a graph showing the fate of raw materials, intermediate products and BH4 when Compound No. 4 was synthesized.

FIG. 8 is a diagram showing a state in which rat GTP cyclohydrolase I purified in Example 6 was digested with lysyl endopeptidase and then eluted with acetonitrile by C18 reverse phase column chromatography.

FIG. 9 shows the strategy used to determine the primary sequence of the rat GTP cyclohydrolase I cDNA.

FIG. 10 is a sequence diagram showing a primary sequence of cDNA of rat GTP cyclohydrolase I and an amino acid sequence deduced from the sequence.

FIG. 11 shows that rat GTP cyclohydrolase I has easy homology with residues in the pterin-binding domain of dihydrofolate reductase.

Continuation of the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 C12N 15/00-15/90 C12P 1/00-41/00 BIOSIS (DIALOG) CA ( STN) REGISTRY (STN) GenBank / EMBL / DDBJ (G ENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. The amino acid sequence from the amino terminal to the 25th amino acid has the formula: Pro-Ser-Leu-Ser-Lys-Glu-Ala-Ala-Leu-Val-His-Glu-Al
a-Leu-Val-Ala-Arg-Gly-Leu-Glu-Thr-Pro-Leu-Arg-Pro, having the ability to convert GTP to D-erythro-7,8-dihydroneopterin triphosphate E. coli-derived GTP cyclohydrolase I. 2. The amino acid sequence up to the 15th amino acid from the amino terminal has the formula: Val-Gly-Leu-Arg-Arg-Arg-Ala-Arg-Leu-Ser-Arg-Leu-Va
D-erythro-7,8-dihydroneopterin triphosphate represented by l-Ser-Phe
-Rat liver-derived 6-pyruvoyltetrahydropterin synthase capable of converting to tetrahydropterin. 3. Partial amino acid sequence: Leu-Leu-Ser-Leu-Leu-Gln-Arg-Asp-Thr-Phe-Gln-Ser-Gl
y-Ala-His-Val-Asp-Phe-Tyr-Asp-Ile, and 6-pyruvoyl-5,6,7,8-tetrahydropterin with 6- (R) -L-erythro-5,6, 7,
Sepiapterin reductase derived from rat erythrocytes, which has the ability to convert to 8-tetrahydrobiopterin.