KR102092075B1 - Crystal structure of MmMVK-mevalonate complex and mevalonate kinase mutants resistant to substrate inhibition - Google Patents

Abstract

The present invention relates to a three-dimensional crystal structure of a Mm MVK-mevalonate complex and a substrate inhibitory resistance muvalonate kinase ( Mm MVK), the inventors of which are Bacillus mevalonate phosphatase proteins and substrates. The three-dimensional structure of the mevalonic acid complex was determined and analyzed by X-ray crystallization. In this three-dimensional structure, a second substrate binding site, which is different from the known substrate binding sites, was found. When mutating the amino acid residue of this new binding site, a mutant protein was found that is resistant to self-inhibition, which inhibits the activity of the enzyme depending on the substrate concentration. The enzyme activity analysis and three-dimensional structure of the mutant protein were determined. The newly developed mutant mevalonic acid phosphatase protein is expected to help improve the yield of isoprene biosynthesis as a rate step enzyme for isoprene biosynthesis in the isoprene biosynthetic pathway, the mevalonic acid pathway.

Description

Three-dimensional crystal structure of MmMVK-mevalonic acid complex and substrate inhibition resistance mutant mevalonic acid phosphatase {Crystal structure of MmMVK-mevalonate complex and mevalonate kinase mutants resistant to substrate inhibition}

The invention Mm MVK- mevalonic acid (mevalonate) 3-dimensional crystal structure and substrate inhibition-resistant mutant kinases mevalonic acid of the complex; relates to (mevalonate kinase Mm MVK).

Isoprene, also known as 2-methyl-1,3-butadiene, is a volatile 5-carbon hydrocarbon. Isoprene is produced by a variety of organisms, including microorganisms, plant and animal species. There are two pathways for isoprene biosynthesis of the mevalonic acid pathway (MVA pathway) and the non-mevalonic acid (or mevalonic acid independent) pathway (MEP pathway) (Figure 1). The MVA pathway is present in the cytosol of eukaryotes, archaea and higher plants. The MEP pathway is found in most bacteria, green algae and chloroplasts of higher plants.

The isoprene compound can be used as a raw material for bio-derived rubber. Isoprene is the final product of the mevalonic acid pathway, a metabolic pathway in which isoprene is biosynthesized from acetylCoA to mevalonic acid, and is used as a precursor to various biomaterials in the future, and is an indispensable substance for bioretention.

Mevalonic acid phosphatase, an enzyme related to phosphorylation of mevalonic acid as an enzyme at a rate step in the mevalonic acid pathway, is caused by feedback inhibition in which enzymatic activity is inhibited by metabolites below mevalonic acid in the isoprene biosynthetic pathway. Became known. However, it has been reported that metabolism of methalonacina mazei, an archaea bacterium, does not cause feedback suppression by a metabolite of mevalonic acid in the isoprene biosynthetic pathway, as previously known (Appl Environ Microbiol. 2011 Nov; 77 (21): 7772-7778).

PCT published patent WO 2006-063752 (2006.06.22 published)

An object of the present invention is Mm MVK- mevalonic acid (mevalonate) 3-dimensional crystal structure and substrate inhibition-resistant mutant kinases mevalonic acid of the complex; to provide a (mevalonate kinase Mm MVK).

In order to achieve the above object, the present invention is a three-dimensional crystal of the Mm MVK-mevalonic acid complex in which a mevalonate kinase ( Mm MVK) and a mevalonate substrate composed of the amino acid sequence of SEQ ID NO: 1 are bound. as a structure, the mevalonic acid (mevalonate) substrate is either coupled to a hinge (hinge) region between the N-terminal domain and the C-terminal domain of Mm MVK, and selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 of Mm MVK Provides a three-dimensional structure determination of the Mm MVK-mevalonic acid complex that binds one or more amino acids.

In addition, the present invention provides a method for screening a substance that promotes the activity of a mevalonate kinase ( Mm MVK) using the three-dimensional structure determination of the Mm MVK-mevalonic acid complex.

In addition, the present invention is a mevalonate kinase ( Mm MVK) consisting of the amino acid sequence of SEQ ID NO: 1, any one or more selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 among the amino acid sequences of SEQ ID NO: 1 Provided is a mutant mevalonic acid phosphatase in which the amino acid residue is substituted with alanine.

In addition, the present invention provides an isoprene production method comprising the step of expressing the mutant mevalonic acid phosphatase in a host cell.

The present invention relates to a three-dimensional crystal structure of a Mm MVK-mevalonate complex and a substrate inhibitory resistance muvalonate kinase ( Mm MVK), the inventors of which are Bacillus mevalonate phosphatase proteins and substrates. The three-dimensional structure of the mevalonic acid complex was determined and analyzed by X-ray crystallization. In this three-dimensional structure, a second substrate binding site, which is different from the known substrate binding sites, was found. When mutating the amino acid residue of this new binding site, a mutant protein was found that is resistant to self-inhibition, which inhibits the activity of the enzyme depending on the substrate concentration. The enzyme activity analysis and three-dimensional structure of the mutant protein were determined. The newly developed mutant mevalonic acid phosphatase protein is expected to help improve the yield of isoprene biosynthesis as a rate step enzyme for isoprene biosynthesis in the isoprene biosynthetic pathway, the mevalonic acid pathway.

1 shows the isoprene biosynthetic pathway.
Figure 2 shows the production process of Mm MVK E. coli expression vector.
Figure 3 shows the results of Mm MVK E. coli mass expression and pure separation purification.
4 shows Mm MVK crystals. Rhombic polyhedron (b) and right-angled polyhedron (c) crystal shapes.
5 shows a diffraction image of Mm MVK crystals.
FIG. 6 shows a diffraction image of a complex crystal obtained by crystallizing crystals of Mm MVK and mevalonate complex as a substrate and AMPPNP complex as a coenzyme ATP analog under similar crystallization conditions.
7 shows the three-dimensional structure of the Mm MVK apo protein.
8 shows the structure of the Mm MVK and mevalonate complex. It can be seen that mevalonate, a substrate, is bound between the N-terminal domain and the C-terminal domain.
9 shows mevalonate bound to the active site. The structure of the mevalonate and the apparent electron density function (Fo-Fc> 3σ), which were originally bound to the active site, were confirmed (right: hydrogen bonds with surrounding residues are indicated by dotted lines). And, it can be seen that it is deeply bound to the surface of the active site (left).
Figure 10 shows the structure of mevalonate bound to the non-active site. The mevalonate bound to the hinge of the N-terminal and C-terminal domains at the back of the active site is represented by an electron density function (Fo-Fc> 2.5σ) (right; dotted for hydrogen bonding, yellow for mevalonate, and MmMVK for green). You can see that it is bound to the back surface of the active site (left).
11 shows a comparison result of a mevalonate (yellow) and apo structure (orange) and a complex (green) structure bound to the second active site. Comparing the structure before and after the mevalonate is bonded, it can be seen that there is a change in the structure of the amino acid residue near the binding site. It can be seen that the amino acid residues Glu295 and Lys292 (indicated by red arrows) were rotated by 130 degrees and 90 degrees, respectively, and moved in the mevalonate direction.
12 shows the hinge center of the domain of the second mevalonate binding site.
13 shows the initial reaction rate of Mm MVK enzyme according to concentration. It can be seen that as the concentration increases, the initial reaction rate decreases at a mevalonate concentration of 15 mM or more.
14 shows amino acid residues at the second substrate binding site. The residues selected for mutagenesis are marked with boxes (red).
15 is Mm MVK It shows the result of the enzyme reaction rate according to the change in the substrate concentration of the mutant protein. As a result of measuring the reaction rate according to the mevalonate concentration of wild type and mutant proteins, it can be seen that the reaction rates of the three mutations 159/292, 295/292 and 159/292/295 did not decrease (above). The highest activity value of the reaction rate and the activity value when the substrate was 50 mM were shown in a bar graph (below).
16 shows the crystallization results in the low- and high-concentration mevalonate conditions of the wild-type Mm MVK protein.
FIG. 17 shows the crystallization results under low and high concentration mevalonate conditions of the mutant K295A / E292A Mm MVK protein.
18 shows a comparison result of the Mm MVK structure. The second substrate binding structure and the high concentration (50 mM substrate) binding complex structure show the largest structural differences.
19 shows the protein surface of Mm MVK 2 ^nd mevalonate structure and Mm MVK + 50 mM mevalonate structure. If you compare active seats It can be seen that the Mm MVK 2 ^nd mevalonate structure is a more open structure than the Mm MVK + 50 mM mevalonate structure. Looking at the difference in surface area, the Mm MVK 2 ^nd mevalonate structure is 426.9 Å ² larger (two figures on the left). When comparing the second mevalonate binding site, it can be seen that the binding site is narrowed in the MmMVK + 50 mM mevalonate structure, so that mevalonate cannot bind (two figures on the right).
20 shows the structural change from the open structure of the Mm MVK to the closed structure. It can be seen that the C-terminal domain overlaps well, but the N-terminal domain is converted into a closed structure by rotating about 10 degrees counterclockwise with respect to the axis of rotation (orange) passing through the fold (green).

The present invention is a three-dimensional crystal structure of an Mm MVK-mevalonic acid complex in which a mevalonate kinase ( Mm MVK) and a mevalonate substrate composed of an amino acid sequence of SEQ ID NO: 1 are bound, wherein the mevalonic acid ( mevalonate) substrate Mm bonded to the hinge (hinge) region between the N-terminal domain and the C-terminal domain of Mm MVK, and in combination with any one or more amino acids selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 of Mm MVK Provides three-dimensional structure determination of MVK-mevalonic acid complex.

Preferably, the mevalonic acid phosphatase consisting of the amino acid sequence of SEQ ID NO: 1 may be derived from Metanosrcina mazei, and may be UniProt ID: Q8PW39, but is not limited thereto.

Preferably, the Mm MVK-mevalonic acid complex has a monoclinic space group of P2 ₁ 2 ₁ 2 ₁ , unit-cell parameters of a = 43.4 Å, b = 96.55 Å , c = 133.71 Å, and α = β = γ = 90 °, but is not limited thereto.

Preferably, the Mm MVK-mevalonic acid complex may form an open structure by a combination of Mm MVK and a mevalonate substrate, but is not limited thereto.

In addition, the present invention provides a method for screening a substance that promotes the activity of a mevalonate kinase ( Mm MVK) using the three-dimensional structure determination of the Mm MVK-mevalonic acid complex.

In addition, the present invention is a mevalonate kinase ( Mm MVK) consisting of the amino acid sequence of SEQ ID NO: 1, any one or more selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 among the amino acid sequences of SEQ ID NO: 1 Provided is a mutant mevalonic acid phosphatase having an amino acid residue substituted with alanine and a functional equivalent thereof.

In detail, the mevalonic acid phosphatase consisting of the amino acid sequence of SEQ ID NO: 1 may be derived from metanosrcina mazei, and may be UniProt ID: Q8PW39, but is not limited thereto.

Specifically, the mutant mevalonic acid phosphatase may be made of SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto. The mutant mevalonic acid phosphatase consisting of SEQ ID NO: 2 is a Lys292 / Glu295 mutant, and the mutant mevalonic acid phosphatase consisting of SEQ ID NO: 3 is a Pro159 / Lys292 / Glu295 mutation.

Specifically, the mutant mevalonic acid phosphatase may have mevalonic acid substrate inhibition resistance.

The "functional equivalent" of the present invention includes all of the amino acid sequence variants in which some or all of the mutant mevalonic acid phosphatases of the present invention are substituted, or a part of the amino acid is deleted or added to maintain the enzyme function. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located in a portion that is not directly involved in the activity of the mutant mevalonic acid phosphatase.

In addition, the present invention provides an isoprene production method comprising the step of expressing the mutant mevalonic acid phosphatase in a host cell.

The step of expressing the mutant mevalonic acid phosphatase in a host cell can be appropriately selected according to techniques known in the art.

Hereinafter, the present invention will be described in detail according to examples not limiting the present invention. It should be understood that the following examples of the present invention are only intended to embody the present invention and do not limit or limit the scope of the present invention. Therefore, from the detailed description and examples of the present invention, what can be easily inferred by experts in the technical field to which the present invention pertains is interpreted as belonging to the scope of the present invention.

< Experimental example >

The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.

1. Protein expression and purification

Methanosarcina mazei ; The gene of mevalonate kinase ( Mm MVK) derived from M. mazei ) is a primer 5'-GCG CAT ATG GTT TCA TGT TCT GCG containing restriction enzymes NdeI and BamHI sites (dark letters) from M. mazei genomic DNA. CCC-3 'and 5'-GCG GGA TCC TCA ATC GAC CTT CAA CCC-3' were amplified by PCR. A 900 bp PCR product was inserted into the expression vector pET-28a (Novagen, Madison, Wisconsin, USA) to express a protein with a histidine tag and a thrombin protease site (MGSSHHHHHHSHSLVLVPRGSH) at the amino terminus. The Mm MVK gene inserted into the expression vector was confirmed by a DNA sequence identification method. The theoretical molecular weight of the protein is about 33.6 kDa and the theoretical isoelectric point is 6.6.

For protein expression, the sequence-identified MmMVK expression vector was transformed into E. coli BL21 (DE3) strain. The transformed E. coli was first cultured overnight in 10 ml of Luria-Bertani (LB) liquid medium, and then inoculated in 1000 ml M9 minimal medium containing antibiotic kanamycin (30 ug / ml), followed by wavelength at 30 degrees Celsius. Incubate until 600 nm absorbance becomes 0.6. And seleno-methionine (Acros Organics, New Jersey, USA) 40 ug / ml and 17 other L-amino acids were added. Then, protein expression was induced at 30 ° C for one day using isopropyl β-D-1 thiogalactopyronoside (IPTG) at a concentration of 0.4 mM. After one day induction, E. coli was harvested by centrifugation (6000 rev / min, 6 min, 4 degrees Celsius). The bacterial mass was evenly mixed with a binding buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol, 1 mM MgCl ₂ ) and then disrupted by ultrasonic waves. After crushing the bacterial solution was centrifuged (12000 rev / min, 1 h), the clear solution of the upper layer was filtered (qualitative filter paper, Advantec, Japan) and poured into a column of nickel-NTA beads. First, the column was washed with 10 column volumes of binding buffer, followed by 20 column volumes of washing buffers (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 30 mM imidazole, 5 mM -mercaptoethanol, 1 mM MgCl ₂ ). Recombinant MmMVK protein was eluted in the column with elution buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 300 mM imidazole, 5 mM -mercaptoethanol, 1 mM MgCl ₂ ). After confirming the portion containing the protein by SDS-PAGE, and collecting and concentrating it, the buffer was 50 mM using an ultrafiltering concentrator Corning Spin-X UF concentrator with 30K molecular-weight cutoff (Corning Inc., New York, USA). Tris-HCl pH 8.5, 1 mM DTT, 1 mM MgCl ₂ was exchanged. And, MmMVK is an anion exchange resin column Resource 15Q column (GE Healthcare, Piscataway, New Jersey, USA) using a linear change to a NaCl concentration of 0-300 mM buffer (50 mM Tris-HCl pH 8.5, 1 mM DTT, 1 mM MgCl ₂ ). After collecting the parts containing MmMVK and concentrating in the same way, finally, gel chromatography using Superdex 200 column (GE Healthcare) in buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM MgCl ₂ ) Separated and purified by graphi. All separation and purification procedures were performed at room temperature using ice-cold buffers believed to stabilize proteins. Protein concentrations were determined by Bradford assay (Bradford, 1976). The histidine tag was not removed for crystallization.

2. Crystallization

Crystallization of MmMVK is initially a screen solution for crystal growth Crystal Screen, Crystal Screen 2, Index (Hampton Research, California, USA), Wizard Screens I and II, Cryo Screens I and II (Emerald BioStructures, Bainbridge Island, Washington, USA) The microbatch crystallization method was performed using a laboratory-made solution at 18 degrees Celsius. 1 μl protein solution (10 mg / ml, 20 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM MgCl ₂ ) and 1 μl screening solution were mixed together and equalized under Al's oil in a 72-well microplate. . First Index Solution No. Protein crystals were made at 82. In order to optimize the protein crystallization conditions, a hanging drop vapor diffusion method was used by varying the concentration of salt and precipitant. The best crystals were obtained under conditions of crystallization solution 0.32 M MgCl ₂ , 0.08 M bis-tris pH 5.5, 16% (w / v) PEG 3350. The droplet size was 3 μl of protein and 3 μl of crystallization solution. For complex crystals with ATP, ADP, AMPPNP, and mevalonic acid, they were immersed in a solution containing 5 mM concentration in apo-MmMVK crystals for 1 to 30 minutes, and then frozen to perform diffraction experiments.

3. X-ray diffraction data collection and data analysis

For X-ray diffraction experiments, the crystals were first immersed in a cryopreservation solution (0.32 M MgCl ₂ , 0.08 M bis-tris pH 5.5, 17% (w / v) PEG 3350, 10% glycerol) for about 10 seconds and then quickly quenched in liquid nitrogen. Soak and freeze. An X-ray diffraction experiment was performed at an absolute temperature of 100 ° C using an ADSC Quantum 210 CCD detector at beamline 5C of the Pohang Accelerator Research Center in Korea, and diffraction data was collected. In order to collect multiple anomalous dispersion (MAD) data, 180 images of each with three wavelengths of X-ray wavelengths 0.97947 Å (peak), 0.97954 Å (edge), and 0.97179 Å (remote) were used, with a vibration angle of 1 degree and an exposure time of 1 second. Data were collected. All diffraction data were analyzed using the HKL-2000 suite (Otwinowski & Minor, 1997) program.

4. Site-directed mutation

MmMVK specific amino acid residues were mutated to alanine according to the producer's method using Stratagene's site-directed mutation kit (Agilten technologies, Inc., Santa Clare, CA, USA). The amino acid residues 159, 292, 293, 295 and 159/293, 159/295, 159, 292, 295/292, and 159/292/295 of MmMVK made single, double and triple mutations, respectively.

5. MmMVK  Active measurement

To measure the activity of the MmMVK enzyme, a 96-well plate was used at 30 degrees Celsius. Each solution contains 0.5 mM PEP, 0.5 mM DTT, 0.32 mM NADH, 10 mM MgCl ₂ , 5 mM ATP, 2 U of LDH, 2 U of PK with 100 μl of 50 mM Tris, 50 mM NaCl pH 7.6 solution have. The enzymatic reaction was initiated by adding 80 nM of purified MmMVK protein. For the measurement of the enzyme reaction, the absorbance at a wavelength of 340 nm was measured to measure the concentration of NADH to the extent of the reaction. The mevalonic acid used for this reaction was mixed with KOH and 1.5: 1 (mol: mol) with KOH, reacted overnight at 37 degrees Celsius, and adjusted to pH 7.0.

6. Structure determination and refinement

The structure of the apo-MmMVK protein was determined by the method of single anomalous dispersion (SAD) using a seleno-methione derivative. The apo-MmMVK high-resolution structure was determined by molecular substitution using the MolRep program of the CCP4 program suite using the structure determined by SAD. The structure was refined with the Phenix program, and the model was built manually with the Coot program. After several refinements and model building, the R-factor is around 20% and the structure is completed when the model no longer improves. The quality of the final model is confirmed by the PROCHECK program. The complex structure of ATP, ADP or mevalonic acid was determined using the apo-MmMVK model by molecular substitution. Structural refinement and model building were performed in the same way.

< Example  1> Mm MVK  E. coli expression vector production, E. coli mass expression and pure separation purification

One. Mm MVK  E. coli expression vector preparation

Methanosarcina mazei of After subcloning mevalonate kinase ( Mm MVK) into the expression vector pET-28a, amplifying the gene by PCR, cutting with restriction enzymes NdeI and BamHI, ligating the vector and transforming it into XL1 (Blue) strain Was selected in LB / Kan medium. The selected strain prepared the vector by DNA mini-prep and confirmed the fragment of the gene inserted in the vector through PCR and restriction enzyme mapping (FIG. 2).

2. Mm MVK  E. coli mass expression and pure purification

The MV protein was purified by pure separation by nickel affinity, ion exchange resin, gel filtration chromatography. The purity of the final isolated protein was confirmed by Native-PAGE and SDS-PAGE (FIG. 3).

< Example  2> Mm MVK  Crystallization and structure determination

One. Mm MVK  Crystallization and X-ray diffraction data collection

Crystallization was obtained by attempting to crystallize the Mm MVK protein at a concentration of 10 mg / ml. Mm MVK protein was crystallized by hanging drop vapor diffusion method under conditions of 16% PEG3350, 0.32 M MgCl ₂ and 0.08 M Bis-Tris pH 5.5. Two crystals were orthogonal and rhombohedral in one solution, but only the rhombic polyhedron was successfully frozen to collect diffraction data (Fig. 4). X-ray diffraction data was collected up to 2.08 Å resolution using a rhombic polyhedron (FIG. 5).

In addition, crystals of Mm MVK and mevalonate complex as a substrate and AMPPNP complex as a coenzyme ATP analog were crystallized under similar determination conditions. High-resolution diffraction data could be obtained from this composite crystal (Fig. 6).

2. Mm MVK  X-ray diffraction data and structure determination of single crystal

(1) Mm MVK apo structure determination and analysis

The three-dimensional structure of Mm MVK was determined by the multiple anomalous dispersion (MAD) method, and the Mm MVK apo three-dimensional structure was modeled and the three-dimensional structure was determined. Finally, the structure of the Mm MVK apo protein was determined at 1.91 Å resolution (Table 1). The final R factor was 25%, and it was confirmed from the Ramachandran plot that 98% of the amino acid residue structure was in an acceptable portion. The Mm MVK protein exists as a dimer, and each protein molecule has a structure in which a beta screen and an alpha helix structure interact (FIG. 7).

Se-Met Native Data peak edge remote Space group P21212 P21212 Unit-cell parameters
(Å) a = 97.11, b = 135.915, c = 46.03,
α = β = γ = 90˚ a = 97.295, b = 135.359, c = 45.867, α = β = γ = 90˚ Distance (mm) 310 280 Exposure time (S) One One Oscillation One One No. of images 180 180 Wavelength (Å) 0.97947 0.97954 0.97179 0.97945 energy (eV) 12658.4 12657.4 12758.4 12658.6 Resolution (Å) 50-2.08
(2.12-2.08) 50-2.08
(2.12-2.08) 50-2.08
(2.12-2.08) 40-1.91
(1.94-1.91) Unique reflections 37136 (1420) 37243 (1019) 38177 (1847) 47072 (1688) Completeness (%) 98.5 (76.8) 97.4 (53.7) 99.7 (98.4) 97.8 (72.2) Rmerge (%) 8.5 (63.3) 7.7 (82.1) 7.4 (0.0) 9.3 (70.9) Redundancy 7.1 (6.2) 7.1 (5.8) 7.1 (5.7) 6. 9 (5.9) I / s (I) 13.7 12.4 10.8 (12.6) 14.4 (13.6) Figure of meritb 0.51 / 0.68 Refinement Resolution (Å) ^c 40.93-1.91
(1.94-1.91) R _work / R _free (%) ^d 25.3 / 30.2 No. of nonhydrogen atoms 4710 No. of water molecules 166 Magnesium ions 2 root mean square deviations Bond length (Å) 0.023 Bond angle (Å) 1.95 Average B factor (Å ² ) Protein 41.523 Water 43.146 Magnesium ions 34.5 Ramachandran plote Most favorable residures (%) 92.5 Additional allowed residues (%) 6.9 Generously allowed residues (%) 0.6

(2) Determination of Mm MVK-mevalonate complex structure

The complex structure of Mm MVK and the substrate mevalonate was determined at a resolution of 1.4 Å resolution (Table 2). In this complex structure, it was confirmed through electron density that the substrate mevalonate was bound to the active site located between the N-terminal domain (1-156) and the C-terminal domain (157-301). In addition, it was found that the second mevalonate is bound to the non-active site (Fig. 8).

Apo MEV complex Data collection Beamline PAL-5C PAL-7A Space group P2 ₁ 2 ₁ 2 P2 ₁ 2 ₁ 2 Unit-cell parameters (Å) a = 97.295, a = 43.4, b = 135.359, b = 96.55, c = 45.867, c = 133.71, α = β = γ = 90 ° α = β = γ = 90˚ Distance (mm) 280 160 Exposure time (S) One One Oscillation One 0.3 No. of images 180 740 Wavelength (Å) 0.97945 0.97933 energy (eV) 12658.6 12660 Resolution (Å) 40-1.91
(1.94-1.91) 50-1.40
(1.42-1.40) Unique reflections 47072 (1688) 110905 (5400) Completeness (%) 97.8 (72.2) 99.4 (98.6) R _merge (%) ^a 9.3 (70.9) 7.4 (70.1) Redundancy 6. 9 (5.9) 8.5 (6.9) I / s (I) 14.4 (13.6) 11.4 Refinement Resolution (Å) ^b 40.9-1.92 26.5 -1.40 R _work / R _free (%) ^c 25.3 / 30.1 20.3 / 23.1 No. of nonhydrogen atoms 4710 5154 No. of water molecules 166 528 magnesium ions 2 2 Mevalonate 3 root mean square deviations Bond length (Å) 0.023 0.028 Bond angle (°) 1.96 2.443 Average B factor (Å ² ) Protein 40.95 20.06 Water 43.13 31.99 Magnesium ions 38.36 21.81 MEV 31.08 Ramachandran plot (%) ^d Residues in favored regions 92.5 98.1 Residues in allowed regions 6.9 1.9 Residues in outlier regions 0.6 0

(3) New mevalonate binding site

In this complex structure, two mevalonates are bound, and it was found that they are bound to the original well-known active site and, specifically, to the back of the original active site rather than the active site (FIGS. 9 and 10). The mevalonate bound to the non-active site was found to be attached to the hinge region where each domain can move to the back of the N-terminal domain and the C-terminal domain (FIGS. 11 and 12).

When overlapping and comparing the structure of the second binding site between Apo and the complex structure, it was found that the structure of two residues, Glu256 and Lys292, was changed by mevalonate binding. The second mevalonate joint of MmMVK is almost identical to the interdoamin hinge, which changes to open and closed structures. Therefore, it is assumed that the second mevalonate bond is correlated with the movement of the hinge.

< Example  3> MmMVK  Mutation of the second substrate binding site And temperament  Structural change and mechanism analysis for inhibition

In the determined Mm MVK and mevalonate substrate complex structure, it was found that a second substrate molecule, which is not a known active site, is bound. In order to analyze the change in the enzyme activity of Mm MVK in the substrate binding of the second binding site, a key amino acid residue is found in this site binding using a site-directed mutation, and a mutant protein of this site is prepared to obtain the Mm MVK enzyme activity. Changes were analyzed.

1. Discovery of the second substrate binding site

Through the structural analysis of Mm MVK and mevalonate, it was found that mevalonate, a substrate, binds to the opposite side of the known binding site. This new mevalonate binding site was thought to be associated with the modulatory action of the Mm MVK enzyme.

2. Mm MVK's  Substrate activity inhibition

The activity of Mm MVK was measured according to the concentration of the substrate. By the way, it was observed that the activity of Mm MVK increases as the concentration of the substrate increases, but when it reaches a certain concentration or more, the activity decreases again (FIG. 13). That is, it can be seen that the activity decreases when the concentration of mevalonate as a substrate becomes 15 mM or more. This indicates that Mm MVK enzyme activity is inhibited due to self-inhibition by the substrate at a specific concentration above the substrate. Analysis of this inhibition of self-activity suggests that inhibition of activity may occur due to the second mevalonate binding, as observed in the three-dimensional structure. It should be changed from the open state to the closed state, but it is thought that the enzyme activity is inhibited because it cannot convert to the closed state by binding of the substrate at the second binding site at a high concentration. For self-inhibition, the substrate binding to the second substrate binding site was thought to be related, and in order to find the correlation, a site-directed mutagenesis of the second binding site amino acid, enzyme activity analysis, and three-dimensional structure study were performed.

3. Mm MVK's  Substrate activity inhibition

Among the amino acid residues interacting with mevalonate at this second substrate binding site, single-single mutations that select Pro159, Lys292, Pro293, and Glu295 to replace with alanine residues, and Pro159Ala Lys292Ala, Pro159Ala Pro293Ala, Pro159Ala E295Ala, Lys292Ala Glu295Ala double mutation and Pro159 Lys292Ala Glu295Ala triple mutations were prepared by a site-directed mutagenesis method, and each mutation was confirmed by reading a vector DNA sequence (FIG. 14). Then, each of these mutant proteins was expressed and purified purely.

4. Enzyme activity analysis of the second substrate binding site mutation

In order to confirm that the second substrate binding site and the enzyme self-inhibition are related, the activity of the enzyme was measured using wild-type protein and each mutant protein (FIG. 15). As can be seen in Figure 15, in the wild type and single mutant, in some double mutants, it can be seen that the initial reaction rate decreases as the concentration of the substrate increases, but the double mutants P159A / K292A, E295A / K292A, and the triple mutants P159A / E295A / K292A showed that the initial reaction rate did not decrease. Therefore, it can be seen from the measurement of the activity of the mutant protein that the second mevalonate binding site is related to the enzyme self-inhibition.

5. In the state of high concentration and low concentration of substrate MmMVK  Structure determination and analysis of enzymes

Mm MVK is expected to have an inhibitory action by a substrate based on the result of a decrease in enzymatic activity at a high substrate concentration (15 mM or more), and in particular, the second substrate binding is expected to be related to the second substrate binding. In order to elucidate the mechanism of activity inhibition of enzymes, a structural analysis study of Mm MVK was performed using low and high concentrations of mevalonate.

(1) Recrystallization of Mm MVK wild type protein

In order to determine the complex structure of the wild-type MmMVK protein with low and high concentrations of mevalonate, complex crystals were made using 5 mM mevalonate at a low concentration and 50 mM mevalonate at a high concentration (FIG. 16).

(2) Crystallization of Mm MVK mutant K295A / E292A protein

In the same way as in the wild type, the mutant protein K295A / E292A protein was made using low and high concentrations of mevalonate to make protein crystals (FIG. 17).

(3) Diffraction data collection of wild type and mutant Mm MVK and low and high concentration mevalonate crystals

A total of 24 diffraction data were collected by performing an X-ray diffraction experiment using wild type and mutant Mm MVK and low and high concentration mevalonate complex crystals. Table 3 shows the diffraction results and statistics.

seWT_Apo WT_5 mM ATP WT_2nd MEV WT_5mM MEV WT_50 mM MEV10 mM AMP 295_5mM MEV Data collection Beamline PAL-5C PAL-5C PAL-7A PAL-7A PAL-7A PAL-7A Space group P2 ₁ 2 ₁ 2 P2 ₁ 2 ₁ 2 _P212121 P2 ₁ 2 ₁ 2 C2 P2 ₁ 2 ₁ 2 Unit-cell parameters (Å) a = 97.11, a = 97.29, a = 43.4, a = 97.27, a = 97.29, a = 97.29, b = 135.92, b = 135.36, b = 96.55, b = 134.01, b = 135.36, b = 135.36, c = 46.03, c = 45.87, c = 133.71, c = 44.61, c = 45.87, c = 45.87, α = β = γ = 90 ° α = β = γ = 90 ° α = β = γ = 90˚ α = β = γ = 90 ° α = β = γ = 90 ° α = β = γ = 90 ° Wavelength (Å) 0.97947 0.97945 0.97933 One One One Resolution (Å) 50-2.08 (2.16-2.08) 40-1.91 (1.94-1.91) 50-1.40 (1.42-1.40) 50-1.64 (1.70-1.64) 50-2.33 (2.42-2.33) 50-2.25 (2.32-2.25) Unique reflections 37064 (3509) 37064 (3504) 110281 (10897) 68913 (1252) 25263 (2477) 28218 (699) Completeness (%) 98.5 (76.8) 99.18 (95.8) 98.9 (98.8) 95.9 (94.6) 99.3 (96.7) 96.1 (93.0) R _merge (%) ^a 8.5 (63.3) 9.3 (70.9) 7.4 (70.1) 62.6 (67.3) 58.9 (86.3) 33.7 (35.6) Redundancy 7.1 (6.2) 6. 9 (5.9) 8.5 (6.9) 10.5 (11.7) 11.1 (9.4) 7.1 (7.2) I / s (I) 13.7 14.4 (13.6) 11.4 248.03 (117.95) 164.58 (14.75) 75.11 (48.58) Refinement Resolution (Å) ^b 45.7-2.08 (2.11 -2 .08) 40.9-1.91 (1.95-1.91) 26.5 -1.40 (1.42-1.39) 33.0-1.64 (1.66-1.64) 37.7-2.33 (2.38-2.33) 33.09-2.25 (2.33-2.25) R _work / R _free (%) ^c 20.12 / 26.87 18.6 / 23.9 18.2 / 20.9 24.54 / 27.79 17.39 / 24.91 18.25 / 25.8 No. of nonhydrogen atoms 4637 4879 5212 4503 4598 4729 No. of water molecules 88 333 553 0 178 191 Cl ^- ions 2 4 - 2 - 4 Mevalonate - - 3 - 2 - root mean square deviations Bond length (Å) 0.007 0.007 0.016 0.007 0.008 0.008 Bond angle (°) 1.05 1.07 1.71 1.1 1.04 1.23 Average B factor (Å ² ) Protein 55.6 44.4 25 27.7 43 37.3 Water 53.5 51.8 37.2 - 43.7 37 Cl ^- ions 54.3 49.2 - 57.6 - 42.6 MEV - - 47.2 - 44.3 - Ramachandran plot (%) ^d Residues in faoured regions 97 98 98 97 98 98 Residues in allowed regions 2.7 2 2 2.3 2 1.8 Residues in outlier regions 0.3 0 0 0.7 0 0.2

(4) Comparison of wild type and mutant Mm MVK with low (5 mM) and high (50 mM) mevalonate complex structures

Looking at each determined structure, mevalonate was not bound in the low-concentration mevalonate crystal structure. It was found that mevalonate is bound in the high-concentration mevalonate complex structure. The binding of ATP-like compounds AMPPNP or ATP could not be seen. The mutant K295A structure was similar to the wild type.

When the Mm MVK apo, Mm MVK + 5 mM ATP, Mm MVK + 5 mM mevalonate, and Mm MVK + 50 mM mevalonate complex structures were overlaid on the Mm MVK 2 ^nd mevalonate structure, the difference in rmsd of the main chain of the structure was shown in Table 4. As shown, the high-concentration substrate complex shows the largest structural difference (FIG. 18).

Mm MVK 2 ^nd overlapping mevalonate structure rmsd value (main chain only comparison) Mm MVK apo 0.538 Å Mm MVK + 5 mM ATP 0.545 Å Mm MVK + 5 mM mevalonate 0.615 Å Mm MVK + 50 mM mevalonate 1.192 Å

(5) Open and closed structures of Mm MVK

Mm MVK 2 ^nd mevalonate structure and Mm MVK + 50 mM as compared to a mevalonate structure, C-terminal domain, but little difference Mm MVK + 50 mM N-terminal domain of the mevalonate structure is more mevalonate than Mm MVK 2 ^nd mevalonate structure It was confirmed that the movement toward the coupling site (Fig. 19).

Looking at these domain movements, the N-terminal domain of the Mm MVK 2 ^nd mevalonate structure is rotated about 9.7 degrees about the axis of rotation passing through the folding residues of amino residues 6-7, 32-33, 85-93, 144-145, 297-298. By the Mm MVK + 50 mM mevalonate structure of the N-terminal domain and overlap was confirmed (Fig. 20).

The Mm MVK 2 ^nd mevalonate structure is an open structure, and the second mevalonate binding site is adjacent to the rotating fold, and it is thought that the mevalonate is bound to the closed structure by binding to this part.

The Mm MVK 2 ^nd mevalonate structure is an open structure, and the Mm MVK + 50 mM mevalonate structure is a closed structure. The open structure has low enzyme activity and the closed structure has high enzyme activity.

Therefore, Mm MVK has high enzymatic activity by converting from an open structure to a closed structure according to the substrate binding. When this structural change does not occur, the enzyme activity is low and the enzyme does not function.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL          UNIVERSITY <120> Crystal structure of MmMVK-mevalonate complex and mevalonate          kinase mutants resistant to substrate inhibition <130> ADP-2018-0394 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 301 <212> PRT <213> Methanosarcina mazei <400> 1 Met Val Ser Cys Ser Ala Pro Gly Lys Ile Tyr Leu Phe Gly Glu His   1 5 10 15 Ala Val Val Tyr Gly Glu Thr Ala Ile Ala Cys Ala Val Glu Leu Arg              20 25 30 Thr Arg Val Arg Ala Glu Leu Asn Asp Ser Ile Thr Ile Gln Ser Gln          35 40 45 Ile Gly Arg Thr Gly Leu Asp Phe Glu Lys His Pro Tyr Val Ser Ala      50 55 60 Val Ile Glu Lys Met Arg Lys Ser Ile Pro Ile Asn Gly Val Phe Leu  65 70 75 80 Thr Val Asp Ser Asp Ile Pro Val Gly Ser Gly Leu Gly Ser Ser Ala                  85 90 95 Ala Val Thr Ile Ala Ser Ile Gly Ala Leu Asn Glu Leu Phe Gly Phe             100 105 110 Gly Leu Ser Leu Gln Glu Ile Ala Lys Leu Gly His Glu Ile Glu Ile         115 120 125 Lys Val Gln Gly Ala Ala Ser Pro Thr Asp Thr Tyr Val Ser Thr Phe     130 135 140 Gly Gly Val Val Thr Ile Pro Glu Arg Arg Lys Leu Lys Thr Pro Asp 145 150 155 160 Cys Gly Ile Val Ile Gly Asp Thr Gly Val Phe Ser Ser Thr Lys Glu                 165 170 175 Leu Val Ala Asn Val Arg Gln Leu Arg Glu Ser Tyr Pro Asp Leu Ile             180 185 190 Glu Pro Leu Met Thr Ser Ile Gly Lys Ile Ser Arg Ile Gly Glu Gln         195 200 205 Leu Val Leu Ser Gly Asp Tyr Ala Ser Ile Gly Arg Leu Met Asn Val     210 215 220 Asn Gln Gly Leu Leu Asp Ala Leu Gly Val Asn Ile Leu Glu Leu Ser 225 230 235 240 Gln Leu Ile Tyr Ser Ala Arg Ala Ala Gly Ala Phe Gly Ala Lys Ile                 245 250 255 Thr Gly Ala Gly Gly Gly Gly Cys Met Val Ala Leu Thr Ala Pro Glu             260 265 270 Lys Cys Asn Gln Val Ala Glu Ala Val Ala Gly Ala Gly Gly Lys Val         275 280 285 Thr Ile Thr Lys Pro Thr Glu Gln Gly Leu Lys Val Asp     290 295 300 <210> 2 <211> 301 <212> PRT <213> Methanosarcina mazei <400> 2 Met Val Ser Cys Ser Ala Pro Gly Lys Ile Tyr Leu Phe Gly Glu His   1 5 10 15 Ala Val Val Tyr Gly Glu Thr Ala Ile Ala Cys Ala Val Glu Leu Arg              20 25 30 Thr Arg Val Arg Ala Glu Leu Asn Asp Ser Ile Thr Ile Gln Ser Gln          35 40 45 Ile Gly Arg Thr Gly Leu Asp Phe Glu Lys His Pro Tyr Val Ser Ala      50 55 60 Val Ile Glu Lys Met Arg Lys Ser Ile Pro Ile Asn Gly Val Phe Leu  65 70 75 80 Thr Val Asp Ser Asp Ile Pro Val Gly Ser Gly Leu Gly Ser Ser Ala                  85 90 95 Ala Val Thr Ile Ala Ser Ile Gly Ala Leu Asn Glu Leu Phe Gly Phe             100 105 110 Gly Leu Ser Leu Gln Glu Ile Ala Lys Leu Gly His Glu Ile Glu Ile         115 120 125 Lys Val Gln Gly Ala Ala Ser Pro Thr Asp Thr Tyr Val Ser Thr Phe     130 135 140 Gly Gly Val Val Thr Ile Pro Glu Arg Arg Lys Leu Lys Thr Pro Asp 145 150 155 160 Cys Gly Ile Val Ile Gly Asp Thr Gly Val Phe Ser Ser Thr Lys Glu                 165 170 175 Leu Val Ala Asn Val Arg Gln Leu Arg Glu Ser Tyr Pro Asp Leu Ile             180 185 190 Glu Pro Leu Met Thr Ser Ile Gly Lys Ile Ser Arg Ile Gly Glu Gln         195 200 205 Leu Val Leu Ser Gly Asp Tyr Ala Ser Ile Gly Arg Leu Met Asn Val     210 215 220 Asn Gln Gly Leu Leu Asp Ala Leu Gly Val Asn Ile Leu Glu Leu Ser 225 230 235 240 Gln Leu Ile Tyr Ser Ala Arg Ala Ala Gly Ala Phe Gly Ala Lys Ile                 245 250 255 Thr Gly Ala Gly Gly Gly Gly Cys Met Val Ala Leu Thr Ala Pro Glu             260 265 270 Lys Cys Asn Gln Val Ala Glu Ala Val Ala Gly Ala Gly Gly Lys Val         275 280 285 Thr Ile Thr Ala Pro Thr Ala Gln Gly Leu Lys Val Asp     290 295 300 <210> 3 <211> 301 <212> PRT <213> Methanosarcina mazei <400> 3 Met Val Ser Cys Ser Ala Pro Gly Lys Ile Tyr Leu Phe Gly Glu His   1 5 10 15 Ala Val Val Tyr Gly Glu Thr Ala Ile Ala Cys Ala Val Glu Leu Arg              20 25 30 Thr Arg Val Arg Ala Glu Leu Asn Asp Ser Ile Thr Ile Gln Ser Gln          35 40 45 Ile Gly Arg Thr Gly Leu Asp Phe Glu Lys His Pro Tyr Val Ser Ala      50 55 60 Val Ile Glu Lys Met Arg Lys Ser Ile Pro Ile Asn Gly Val Phe Leu  65 70 75 80 Thr Val Asp Ser Asp Ile Pro Val Gly Ser Gly Leu Gly Ser Ser Ala                  85 90 95 Ala Val Thr Ile Ala Ser Ile Gly Ala Leu Asn Glu Leu Phe Gly Phe             100 105 110 Gly Leu Ser Leu Gln Glu Ile Ala Lys Leu Gly His Glu Ile Glu Ile         115 120 125 Lys Val Gln Gly Ala Ala Ser Pro Thr Asp Thr Tyr Val Ser Thr Phe     130 135 140 Gly Gly Val Val Thr Ile Pro Glu Arg Arg Lys Leu Lys Thr Ala Asp 145 150 155 160 Cys Gly Ile Val Ile Gly Asp Thr Gly Val Phe Ser Ser Thr Lys Glu                 165 170 175 Leu Val Ala Asn Val Arg Gln Leu Arg Glu Ser Tyr Pro Asp Leu Ile             180 185 190 Glu Pro Leu Met Thr Ser Ile Gly Lys Ile Ser Arg Ile Gly Glu Gln         195 200 205 Leu Val Leu Ser Gly Asp Tyr Ala Ser Ile Gly Arg Leu Met Asn Val     210 215 220 Asn Gln Gly Leu Leu Asp Ala Leu Gly Val Asn Ile Leu Glu Leu Ser 225 230 235 240 Gln Leu Ile Tyr Ser Ala Arg Ala Ala Gly Ala Phe Gly Ala Lys Ile                 245 250 255 Thr Gly Ala Gly Gly Gly Gly Cys Met Val Ala Leu Thr Ala Pro Glu             260 265 270 Lys Cys Asn Gln Val Ala Glu Ala Val Ala Gly Ala Gly Gly Lys Val         275 280 285 Thr Ile Thr Ala Pro Thr Ala Gln Gly Leu Lys Val Asp     290 295 300

Claims (10)

A three-dimensional crystal structure of the Mm MVK-mevalonic acid complex in which a mevalonate kinase ( Mm MVK) and a mevalonate substrate composed of the amino acid sequence of SEQ ID NO: 1 are bound,
The mevalonic acid (mevalonate) substrate Mm MVK of the N-terminal domain and the C-terminal coupling of a hinge (hinge) region between the domains, and any one or more amino acids selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 of Mm MVK And, the Mm MVK-mevalonic acid complex has an orthorhombic space group of P2 ₁ 2 ₁ 2 _1, and a unit-cell parameter of a = 43.4 Å, b = 96.55 Å. , c = 133.71 ,, α = β = γ = 90 ° Mm MVK- mevalonic acid complex three-dimensional crystal structure characterized in that. According to claim 1, wherein the mevalonic acid phosphatase consisting of the amino acid sequence of SEQ ID NO: 1 Mm MVK-mevalonic acid complex three-dimensional structure determination, characterized in that derived from metanosrcina mazei (Methanosarcina mazei). delete According to claim 1, wherein said Mm MVK- mevalonic acid mevalonic acid complexes Mm MVK- complex three-dimensional structure determination as to form an open frame by the engagement of Mm MVK and mevalonic acid (mevalonate) substrate. A method for screening a substance that promotes the activity of a mevalonate kinase ( Mm MVK) using the three-dimensional structure determination of the Mm MVK-mevalonic acid complex according to claim 1, 2, or 4. In the mevalonate kinase ( Mm MVK) consisting of the amino acid sequence of SEQ ID NO: 1, alanine (Alanine) any one or more amino acid residues selected from the group consisting of Pro159, Lys292, Pro293 and Glu295 among the amino acid sequences of SEQ ID NO: 1 alanine). The method of claim 6, wherein the mevalonic acid phosphatase consisting of the amino acid sequence of SEQ ID NO: 1 is a mutant mevalonic acid phosphatase, characterized in that derived from metanosrcina mazei (Methanosarcina mazei). The mutant mevalonic acid phosphatase according to claim 6, characterized in that it consists of SEQ ID NO: 2 or SEQ ID NO: 3. 7. The mutant mevalonic acid phosphatase according to claim 6, wherein the mutant mevalonic acid phosphatase is a mevalonic acid substrate resistance resistant mutation. A method for producing isoprene, comprising expressing the mutant mevalonic acid phosphatase according to any one of claims 6 to 9 in a host cell.

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