CgMetK protein crystal structure from Corynebacterium glutamicum and uses thereof

Abstract

The present invention relates to the crystal structure of the CgMetK protein, which is an S-adenosylmethionine synthetase (MetK) derived from Corynebacterium glutamicum, and its uses. Specifically, the crystal structures of the apo form and the adenosine / triphosphate complex form of CgMetK were identified. As a result, it was shown that CgMetK has an allosteric inhibitory binding site for SAM products near the active site, which is inhibited competitively and non-competitively by SAM. Through structure-guided protein engineering, a CgMetKE68A variant was developed that exhibits near-complete inhibition of SAM-mediated release along with enhanced enzyme activity. Crystal structure analysis of the CgMetKE68A variant revealed that the formation of novel hydrogen bonds between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and also improved protein thermal stability by enhancing enzyme tetramerization.

Description

Technology Field

[0001] The present invention relates to Corynebacterium glutamicum ( Corynebacterium glutamicum S-adenosylmethionine synthetase (MetK) derived from ) Cg This relates to the crystal structure of the MetK protein and its applications. Background Technology

[0002] S-adenosylmethionine (SAM) is a compound essential to most living organisms, functioning as a biological methyl donor. It is used in the synthesis of various biomolecules, such as proteins, DNA, RNA, and phospholipids. Several studies suggest that SAM regulates cell growth through nucleotide methylation. Furthermore, it regulates numerous biosynthetic pathways, such as methionine biosynthesis, polyamine biosynthesis, and antibiotic biosynthesis. Since SAM is essential to all organisms, metK is an essential gene. metK Fruiting strains have other functional properties metK It was revealed that survival is possible only when genes are present.

[0003] The synthesis of SAM is catalyzed by MetK in a two-step reaction. First, S with methionine and the adenosine moiety of ATP N SAM is formed by reaction 2, and subsequently, triphosphate is broken down into pyrophosphoric acid and orthophosphoric acid, releasing the final products (Fig. 1A). Most ATP-dependent enzymes carry out irreversible reactions through the high-energy phosphate binding of ATP, and MetK similarly carries out an irreversible reaction. To enhance enzyme activity, MetK contains Mg 2 + Divalent metal ions such as and K + It requires monovalent metal ions such as.

[0004] Corynebacterium glutamicum ( Corynebacterium glutamicum It is generally a GRAS-rated organism and is commonly used in the food industry to produce amino acids such as lysine and glutamate. Additionally, to increase productivity or produce high-value compounds such as biofuels C . glutamicum Extensive research on metabolic engineering has been conducted. Most of the applied metabolic engineering strategies have been genome editing, such as the overexpression or introduction of useful genes. Nevertheless, these strategies are inefficient in situations where enzyme activity is inhibited; conversely, protein engineering can address this problem instead. Although there are results showing that MetK is inhibited by SAM, C . glutamicum Origin MetK( Cg Adequate research on enzyme inhibition and protein engineering of MetK has not been sufficiently conducted to date. Prior art literature

[0005] Chinese Patent Publication CN 110172454 A (Published Aug 27, 2019) The problem to be solved

[0006] The present invention relates to Corynebacterium glutamicum ( Corynebacterium glutamicum ) derived S-adenosylmethionine synthetase (MetK) protein ( Cg We intend to provide a crystal of MetK and a method for crystallizing the same.

[0007] In addition, the present invention has reduced enzyme activity inhibition by SAM and increased thermal stability. Cg Provides MetK variants.

[0008] In addition, the present invention is said to CgWe intend to provide a polynucleotide encoding a MetK variant, a recombinant vector containing said polynucleotide, and a microorganism transformed with said recombinant vector.

[0009] In addition, the present invention comprises a step of culturing the microorganism, wherein the inhibition of enzyme activity by SAM is reduced and thermal stability is increased. Cg We intend to provide a method for producing MetK variants.

[0010] In addition, the present invention is said to Cg The present invention aims to provide a crystal of a MetK variant and a method for crystallizing the same.

[0011] In addition, the present invention is said to Cg MetK crystals or Cg Using the crystalline stereostructure of MetK variants Cg We intend to provide a screening method for MetK activity modifiers. means of solving the problem

[0012] To solve the above problem, the present invention comprises a space group of C222, unit cell parameters of a = 129.15 Å, b = 203.89 Å, c = 144.19 Å, α = β = γ = 90°, and an amino acid sequence represented by SEQ ID NO. 1. Cg Provides a crystal of MetK.

[0013] In addition, the present invention comprises i) a reservoir solution comprising 10 to 50% (v / v) polyethylene glycol (PEG) 200, 0.01 to 1M MES with a pH of 6.0 to 8.0, and 1 to 10% (v / v) PEG 3,000, and ii) Cg A protein solution containing MetK protein comprising the step of mixing the protein solutions together and crystallizing them by a vapor diffusion method, having the following crystal form CgA method for crystallizing MetK is provided.

[0014] [Decision form]

[0015] The space group is C222, the unit cell parameters are a = 129.15 Å, b = 203.89 Å, c = 144.19 Å, and α = β = γ = 90°.

[0016] In addition, the present invention comprises an amino acid sequence in which glutamic acid (E), the 68th amino acid in the amino acid sequence represented by SEQ ID NO. 1, is substituted with alanine (A), wherein the inhibition of enzyme activity by S-adenosylmethionine (SAM) is reduced and thermal stability is increased. Cg Provides MetK variants.

[0017] In addition, the present invention is said to Cg A polynucleotide encoding a MetK variant, a recombinant vector comprising said polynucleotide, and a microorganism transformed with said recombinant vector are provided.

[0018] In addition, the present invention comprises the step of culturing the microorganism; and from the cultured microorganism, the Cg A method comprising the step of obtaining a MetK variant, wherein enzyme activity inhibition by SAM is reduced and thermal stability is increased Cg A method for producing MetK variants is provided.

[0019] In addition, the present invention comprises a space group of C121, unit cell parameters of a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, α = γ = 90°, β = 128.91°, and an amino acid sequence represented by SEQ ID NO. 2. Cg Provides a crystal of the MetK variant.

[0020] In addition, the present invention comprises i) a reservoir solution comprising 10 to 50% (v / v) PEG 20,000 and 0.01 to 1M MES with a pH of 6.0 to 8.0, and ii) an amino acid sequence represented by SEQ ID NO. 2. Cg A crystalline form having the following, comprising the step of mixing protein solutions containing MetK variant proteins together and crystallizing them by a vapor diffusion method. Cg A method for crystallizing MetK variants is provided.

[0021] [Decision form]

[0022] The space group is C121, the unit cell parameters are a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, and α = γ = 90°, β = 128.91°.

[0023] In addition, the present invention is said to Cg MetK crystal or the above Cg Using the crystalline stereostructure of the MetK variant Cg MetK activity regulating candidate peptides or Cg A step of generating or screening MetK binding candidate compounds; and ii) the candidate peptide or compound generated or screened in step i) Cg A step including determining whether it regulates MetK activity, Cg A screening method for MetK activity modifiers is provided. Effects of the invention

[0024] The present invention relates to Corynebacterium glutamicum ( Corynebacterium glutamicum S-adenosylmethionine synthetase (MetK) derived from ) Cg This relates to the crystal structure of the MetK protein and its uses, specifically. CgThe crystal structures of the apo and adenosine / triphosphate complex forms of MetK were confirmed. As a result, Cg MetK possesses an allosteric inhibitory binding site for SAM products near its active site, indicating that it is inhibited competitively and non-competitively by SAM. Through structure-guided protein engineering, it demonstrates near-complete inhibition of release by SAM, along with enhanced enzymatic activity. Cg MetK E68A A variant was developed. Cg MetK E68A Crystal structure analysis of the variants revealed that the formation of novel hydrogen bonds between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and improved protein thermal stability by enhancing enzyme tetramerization. Brief explanation of the drawing

[0025] Fig. 1 is Cg This shows the monomer structure of MetK. Figure 2 is Cg This shows the dimerization and tetramerization modes of MetK. Fig. 3 is Cg Represents the active site of MetK. Fig. 4 is Cg This represents the allosteric sites of MetK. Figure 5 is for inhibition reduction Cg This shows the rational protein engineering results of MetK. Specific details for implementing the invention

[0026] The inventors of the present invention Cg The structure and biochemical properties of MetK were analyzed. As a result, CgIt was shown that MetK has an allosteric inhibitory binding site for SAM products near its active site, and that its enzymatic activity is inhibited competitively and non-competitively by SAM. Furthermore, the inventors dramatically reduced inhibition by SAM through rational protein engineering. Cg MetK E68A A variant was developed, and the present invention was completed.

[0027] The present invention comprises a space group of C222, unit cell parameters of a = 129.15 Å, b = 203.89 Å, c = 144.19 Å, α = β = γ = 90°, and an amino acid sequence represented by SEQ ID NO. 1. Cg Provides a crystal of MetK.

[0028] In addition, the present invention comprises i) a reservoir solution comprising 10 to 50% (v / v) polyethylene glycol (PEG) 200, 0.01 to 1M MES with a pH of 6.0 to 8.0, and 1 to 10% (v / v) PEG 3,000, and ii) Cg A protein solution containing MetK protein comprising the step of mixing the protein solutions together and crystallizing them by a vapor diffusion method, having the following crystal form Cg A method for crystallizing MetK is provided.

[0029] [Decision form]

[0030] The space group is C222, the unit cell parameters are a = 129.15 Å, b = 203.89 Å, c = 144.19 Å, and α = β = γ = 90°.

[0031] In the present invention, the terms "enabling crystallization" or "possessing crystallinity" refer to forming solid particles of a specific shape and size from a homogeneous liquid phase or further stabilizing the crystalline state of a protein by utilizing methods such as introducing mutations into protein molecules to make the protein suitable for X-ray protein three-dimensional structure analysis. The three-dimensional structure of a protein is very important for understanding its in vivo function.

[0032] In the present invention, the term "space group" refers to the symmetry of a unit cell of a crystal, wherein a group is formed by combining symmetry elements. The term "space group" may be used interchangeably with "space group."

[0033] In the present invention, the vapor diffusion method includes a sitting drop vapor diffusion method or a hanging-drop vapor diffusion method, and specifically, it may be performed by a sitting drop vapor diffusion method, but is not limited thereto.

[0034] In the above vapor diffusion method, a protein solution is brought into equilibrium with a large aqueous reservoir solution containing a precipitating agent at a concentration suitable for producing crystals. Typically, a purified protein solution is mixed with an equal amount of the reservoir solution so that the concentration of the precipitating agent is about half that required for crystallization, and this solution is suspended under a coverslip that seals the top of the reservoir or attached to the top of a container. Then, the sealed container is left for 1 day to 1 year, usually 2 to 6 weeks, to allow the crystals to grow. Specifically, the vapor diffusion method is a crystallization method that utilizes the fact that when a small drop of mother liquor and a much larger reservoir solution coexist in a sealed space while separated, the movement of water or other volatile substances occurs between them, resulting in a supersaturated state under the protein solution conditions, and that the protein precipitates in response to changes in the precipitating agent in such a thermodynamic metastable state. When proteins precipitate, the process proceeds slowly to reach a stable crystallization state, and the precipitating agent plays a role in reducing the solubility of the concentrated protein solution. To reduce the relative adsorption layer around the protein molecules, the proteins gather together and form crystals.

[0035] The reservoir solution contains precipitating agents, buffers, salts, and detergents mixed at various concentrations. Typically, the protein solution and these reservoir solutions under different conditions are mixed in a 1:1 ratio to form droplets, which are then placed and sealed. Initially, the protein concentration within the droplet differs from that of the reservoir solution, so the protein does not exist in a crystalline state. When left in this sealed state, equilibrium is gradually reached, and crystals are formed under specific conditions based on the principle described above. In particular, the droplet mixed vapor diffusion method provides crystals of a size sufficient for analyzing protein structures. In the droplet mixed vapor diffusion method, a reagent containing the sample and a pure liquid reagent are attached to the top of a container in a state of vapor equilibrium. To allow the sample, which has a lower reagent content relative to the container, to reach equilibrium, water contained in the sample is allowed to drip into the container. This process removes the water from the sample until its concentration becomes equal to that of the liquid reagent, resulting in the acquisition of protein crystals that have reached equilibrium.

[0036] In such vapor diffusion methods, not only the precipitating agent in the reservoir solution but also the types and optimal concentrations of salts, buffers, and surfactants, as well as the pH of the solution and the experimental temperature, are selected differently depending on the type of protein and, in some cases, become very important factors in the crystal formation of the protein.

[0037] In addition, the present invention comprises an amino acid sequence in which glutamic acid (E), the 68th amino acid in the amino acid sequence represented by SEQ ID NO. 1, is substituted with alanine (A), wherein the inhibition of enzyme activity by S-adenosylmethionine (SAM) is reduced and thermal stability is increased. Cg Provides MetK variants.

[0038] In addition, the present invention is said to Cg Provides a polynucleotide encoding a MetK variant.

[0039] The above “polynucleotide” is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless specifically noted otherwise.

[0040] The above polynucleotide is the above Cg It includes not only the nucleotide sequence encoding the MetK variant, but also a sequence complementary to that sequence. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence.

[0041] Additionally, the polynucleotide may be modified. Such modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is interpreted to also include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence. Such substantial identity may be a sequence that exhibits at least 80% homology, at least 90% homology, or at least 95% homology when the nucleotide sequence is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0042] In addition, the present invention provides a recombinant vector comprising the above polynucleotide.

[0043] In addition, the present invention provides a microorganism transformed with the recombinant vector.

[0044] In the present invention, “vector” means a self-replicating DNA molecule used to carry a clonal gene (or another piece of clonal DNA).

[0045] In the present invention, “recombinant vector” means a plasmid, viral vector, or other medium known in the art capable of expressing a nucleic acid inserted within a host cell, and may be a polynucleotide encoding the peptide of the present invention operably linked to a conventional expression vector known in the art. The recombinant vector may generally comprise a replication origin capable of proliferating in a host cell, one or more expression regulatory sequences (e.g., promoter, enhancer, etc.) that regulate expression, a selective marker, and a polynucleotide encoding the peptide of the present invention operably linked to the expression regulatory sequence. The transformed microorganism may be transformed by the recombinant vector.

[0046] Preferably, the transformed microorganism of the present invention Cg A recombinant vector containing a polynucleotide encoding a MetK variant can be obtained by introducing it into a host cell by methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into a cell.

[0047] Any host cell known in the art can be used to introduce the above vector, for example, strains of the genus Bacillus such as E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus churingensis, as well as intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0048] In addition, the present invention comprises the step of culturing the microorganism; and from the cultured microorganism, the Cg A method comprising the step of obtaining a MetK variant, wherein enzyme activity inhibition by SAM is reduced and thermal stability is increased Cg A method for producing MetK variants is provided.

[0049] In addition, the present invention comprises a space group of C121, unit cell parameters of a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, α = γ = 90°, β = 128.91°, and an amino acid sequence represented by SEQ ID NO. 2. Cg Provides a crystal of the MetK variant.

[0050] In addition, the present invention comprises i) a reservoir solution comprising 10 to 50% (v / v) PEG 20,000 and 0.01 to 1M MES with a pH of 6.0 to 8.0, and ii) an amino acid sequence represented by SEQ ID NO. 2. Cg A crystalline form having the following, comprising the step of mixing protein solutions containing MetK variant proteins together and crystallizing them by a vapor diffusion method. Cg A method for crystallizing MetK variants is provided.

[0051] [Decision form]

[0052] The space group is C121, the unit cell parameters are a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, and α = γ = 90°, β = 128.91°.

[0053] In addition, the present invention is said to Cg MetK crystal or the above Cg Using the crystalline stereostructure of the MetK variant Cg MetK activity regulating candidate peptides or Cg A step of generating or screening MetK binding candidate compounds; and ii) the candidate peptide or compound generated or screened in step i) Cg A step including determining whether it regulates MetK activity, Cg A screening method for MetK activity modifiers is provided.

[0054] The present invention is described in detail below according to embodiments that do not limit the invention. It should be understood that the following embodiments of the present invention are merely for the purpose of embodying the invention and do not limit or restrict the scope of the rights of the present invention. Accordingly, anything that can be easily inferred by a person skilled in the art to which the present invention pertains from the detailed description and embodiments of the present invention is interpreted as falling within the scope of the rights of the present invention.

[0055] <Experimental Example>

[0056] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0057] 1. Cg MetK's Cloning , expression and purification

[0058] C. glutamicum origin metKThe gene (UniProtKB accession code: Q9K5E4) was cloned into a pET30a expression vector, and the resulting expression vector is Escherichia coli It was transformed into strain BL21 (DE3)-T1R. E. coli The strain was cultured in 1 L of Luria-Bertani broth containing 50 mg / L kanamycin at 37°C to an optical density of 0.6 at 600 nm. Next, 0.5 mM isopropyl β- D Induction was performed by adding -1-thiogalactopyranoside. After induction overnight at 18°C, the grown cells were harvested by centrifuging at 4,000 g for 15 minutes at 20°C. The cell pellet was resuspended in buffer A (40 mM Tris-HCl, pH 8.0) and then disrupted by sonication. Cell debris was removed by centrifuging at 13,000 g for 30 minutes at 4°C, and the supernatant was applied to Ni-NTA agarose resin (Thermo Fisher Scientific, USA). After washing with buffer B (40 mM Tris-HCl and 10 mM imidazole, pH 8.0), the bound proteins were eluted with buffer C (40 mM Tris-HCl and 300 mM imidazole, pH 8.0). Finally, trace contaminants were removed by size chromatography using a Hiprep 26 / 60 Sephacryl S-300 HR column (GE Healthcare, USA) equilibrated with buffer A. All purification experiments were performed at 4°C. The purified proteins were concentrated to 38 mg / ml in buffer A. Site-targeted mutation experiments were performed using a Quick-Change kit (Agilent, USA), and the mutant proteins were wild-type Cg It was produced using the same protocol used in MetK.

[0059] 2. Cg MetK's crystallization

[0060] Refined Cg MetK was initially crystallized at 20°C by a sitting drop vapor diffusion method using commercially available sparse-matrix screens from Rigaku and Molecular Dimensions. Cg MetK was crystallized in 30% (v / v) polyethylene glycol 200, 0.1 M MES pH 6.0, and 5% (w / v) polyethylene glycol 3,000. Cg For the co-crystallization of MetK and the substrate, Cg MetK was reacted with MgCl2 and KCl at a 20-fold concentration of protein, and with ATP and methionine at a 10-fold concentration of protein at 4°C. After reacting overnight, the reacted proteins were undenatured Cg It was crystallized using the same protocol used for MetK. Co-crystallized with MgCl2, KCl, ATP, and methionine Cg MetK was crystallized in 20% (w / v) polyethylene glycol 3,350 and 0.2 M sodium phosphate monobasic monohydrate. Purified Cg MetK E68A The variant is unmodified Cg It was crystallized using the same protocol used in MetK. Cg MetK E68A The variant was determined using 12% (w / v) polyethylene glycol 20,000 and 0.1 M MES pH 6.5.

[0061] The above crystal was transferred to an antifreeze solution composed of 25% (v / v) glycerol and rapidly frozen directly in liquid nitrogen.

[0062] 3. Cg MetK's X- ray Data Collection and Structure Determination

[0063] Cg X-ray diffraction data of MetK crystals were collected using a Quantum 270 CCD detector (ADSC, USA) at the 7A beamline of the Pohang Accelerator Laboratory (PAL, Republic of Korea). All data were indexed, integrated, and expanded using the HKL-2000 software package. Cg The MetK apo crystal was diffracted with a resolution of 2.2 Å and belonged to space group C3, with unit cell parameters of a = 129.15 Å, b = 203.89 Å, c = 144.19 Å, and α = β = γ = 90°. 3 per asymmetric unit Cg It has a MetK molecule, and the Matthews coefficient is 3.59 Å. -3 Da - 1 It was, which corresponds to a solvent content of about 65.73%. Cg The structure of MetK was determined using the structure of MetK derived from Mycobacterium tuberculosis (PDB code: 3TDE) by performing molecular substitution with MOLREP software in the CCP4 suite. Model construction was performed manually using WinCoot, and purification was performed using Refmac5 software in the CCP4 suite. It formed a complex with MgCl2, KCl, ATP, and methionine. Cg The MetK crystal was diffracted at 2.4 Å and belonged to space group P21 with unit cell parameters of a = 61.311 Å, b = 116.81 Å, c = 116.31 Å, and α = γ = 90°, β = 103.31°. Four complexes formed per asymmetric unit Cg It has a MetK molecule, and the Matthews coefficient is 2.30 Å. -3 Da - 1It was, which corresponds to a solvent content of approximately 46.50%. Formed a complex Cg The structure of MetK was determined using the apo structure by performing molecular substitution with the MOLREP software in the CCP4 suite. Model construction was performed manually using WinCoot, and refinement was performed using the Refmac5 software in the CCP4 suite. Cg MetK E68A The variant crystal was diffracted at a resolution of 1.7 Å and belonged to space group C121 with unit cell parameters of a = 150.4 Å, b = 125.44 Å, c = 116.31 Å and α = γ = 90°, β = 128.91°. Four complexes formed per asymmetric unit Cg MetK E68A It has a molecule, and the Matthews coefficient is 2.42 Å. -3 Da - 1 This corresponds to a solvent content of approximately 49.20%. Model construction was performed manually using WinCoot, and purification was performed using Refmac5 software from the CCP4 suite. Data statistics are summarized in Table 1. apo form, complex form, and E68A variant Cg The MetK structures were deposited in the Protein Data Bank with PDB codes of 8JZH, 8JZG, and 8JZI, respectively.

[0064] Cg MetK Apo Cg MetK Complex Cg MetK E68A Data collection Space group C222 P21 C2 Cell dimensions a , b , c (Oh) 129.1, 203.8, 144.1 61.31, 116.8, 116.3 150.4, 125.4, 116.3 α, β, γ(°) 90, 90, 90 90, 103.31, 90 90, 128.91, 90 Resolution (Å) 50.00 ~ 2.20 50.00 ~ 2.40 50.00 ~ 1.76 R sym or R merge 7.3 (30.0) 6.7 (38.3) 48.4 (48.4) I / s ( I ) 38.3 (5.75) 20.7 (3.38) 36.5 (27.4) Completeness (%) 99.0 (98.8) 99.3 (99.1) 99.4 (94.9) Redundancy 5.9 (5.8) 3.6 (3.2) 3.7 (3.1) Refinement Resolution (Å) 50.0-2.20 50-2.40 50.00 ~ 1.76 No. reflections 90568 59022 156528 R work / R free 22.2 / 26.2 19.5 / 27.0 19.7 / 22.9 No. atoms 9052 11893 12386 Protein 8861 11588 11588 Ligand / ion 12 240 56 Water 179 65 742 B -factors 38.2 39.2 19.2 Protein 39.9 42.9 20.3 Ligand / ion 46.8 43.2 31.9 Water 38.5 29.0 26.0 Rms deviations Bond lengths (Å) 0.0108 0.0099 0.0111 Bond angles (°) 1.7343 1.7686 1.7582

[0065] 4. Cg MetK's In vitro enzyme activity analysis

[0066] Cg To measure the activity of MetK, CgMalachite green assay was used to detect phosphates produced by MetK. The assay was performed at room temperature with a 120-µl reaction volume containing 0.1 M Tris-HCl pH 9.0, various concentrations of methionine (0.01–2 mM), ATP (0.1–5 mM), and 1 μM protein. After the reaction was initiated following the addition of the enzyme, the inventors mixed 180 μl of 1.05% (w / v) ammonium molybdate, 0.03375% (w / v) malachite green, and 1 N HCl into the 20 µl reaction mixture every minute. The enzyme assay was performed in three replicates, and the absorbance of the mixture was measured at 620 nm using a 96-well plate reader. To evaluate the inhibitory effect of SAM, similar enzyme assays were performed by adding various concentrations of SAM to the reaction mixture.

[0067] 5. High-pressure size exclusion chromatography

[0068] Cg To confirm the monomeric state of MetK, High Pressure Size Exclusion Chromatography (HPSEC) was performed using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, USA) equilibrated with 40 mM Tris-HCl pH 8.0 and 150 mM NaCl. 1 ml of the protein sample was injected at a concentration of 1 mg / ml. Calibration curves were generated by injecting ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa) (GE Healthcare, USA) as standard samples.

[0069] 6. Cg MetK's Melting temperature measurement

[0070] Cg To confirm the thermal stability of Met, the inventors measured melting curves using a fluorescence-based protein thermal shift dye kit (Applied Biosystems, USA) and StepOnePlus Real-Time PCR (Thermo Fisher Scientific, USA). The reaction mixture contained 5 μg protein, 0.1 M Na2HPO4-HCl pH 7.0, and 1X protein thermal shift dye. As the temperature increased from 25°C to 99°C, the signal changed due to protein denaturation. Melting temperature ( T m ) was calculated using the first derivative curve.

[0071] < Examples 1> Cg MetK's Overall structure

[0072] The inventors [in order] to investigate molecular mechanisms Cg The crystal structure of MetK was confirmed at a resolution of 2.2 Å (Table 1). Cg The crystal structure of MetK belonged to the C222 space group, and there were three molecules in the asymmetric unit. Cg The overall structure of MetK has RMSD deviations of 0.899, 0.768, and 0.816 Å, respectively. Mycobacterium tuberculosis ( Mt MetK, PDB code: 3TDE), Escherichia coli ( Ec MetK, PDB code: 1P7L) and Thermus thermophilus ( Tt It was similar to other microbial-derived MetKs such as MetK (PDB code: 5H9U) (Fig. 1B). CgThe monomer structure of MetK consisted of three different domains: an N-terminal domain (NTD), a central domain (CD), and a C-terminal domain (CTD) (Fig. 1C). The NTD consisted of two α-helices (α3 and α4), one η-helice (η1), and four β-strands (β1, β6, β7, and β8). The CD consisted of two α-helices (α1 and α2) and three β-strands (β2, β3, and β4). The CTD consisted of four α-helices (α5, α6, α7, and α8), two η-helices (η2 and η3), and three β-strands (β5, β9, and β10) (Fig. 1D). Through the present invention, it was shown that the structure has an invisible region (Gln103-Glu125) near the substrate binding pocket within the CD, and it was estimated that the region may undergo an ordered-disordered conformational change upon substrate binding.

[0073] < Examples 2> Cg MetK's monomer state

[0074] Although there are three molecules in the asymmetric unit of the above crystal structure, Cg The tetrameric state of MetK was observed by C222 crystallographic symmetry operation. In solution Cg In order to confirm the monomeric state of MetK, the inventors performed high pressure size-exclusion chromatography (HPSEC), wherein the above Cg The MetK protein was eluted in the form of a tetramer with a molecular weight of approximately 173.2 kDa (Fig. 2A). These results CgThis indicates that MetK exists as a tetramer in solution. When the inventors calculated the dimerization surface and interactions using PDBePISA, the average area of ​​the dimerization surface was 3,163.7 Å. 2 was. By interacting with Arg8, Asp191, Met274, Arg276, Tyr331, and Glu333 Cg MetK forms a dimer. The side chain of Arg8 interacts with the side chain of Asp191 and the side chains of neighboring monomer-derived Tyr331 and Glu333. The main chain of Met274 interacts with the side chain of neighboring monomer-derived Arg276 (Figs. 2B and 2C). In the dimer, the positions of the chains are almost perfectly 180° axially symmetric with respect to each other, so the residues involved in hydrogen bonding and salt bridging in each chain are identical. Tetramerization involves two Cg It is formed between MetK dimers (Fig. 2B). The average area of ​​the tetramerization surface is 1,740.8 Å. 2 It was calculated as follows. Hydrogen bonds are formed between the main chains of Val96 and Val98, between the side chains of Ser97 and Ser99, and between the side chain and the main chain of Ser85, respectively (Fig. 2D). Similar to dimerization, since the positions of the chains are almost perfectly 180° axially symmetric with respect to each other, the residues involved in tetramerization are the same in the tetramer.

[0075] < Examples 3> Cg MetK's Active site

[0076] Cg To determine the binding mode of metal ions and substrates in MetK, the inventors [identified] Mg 2+ and K + metal ions, methionine, and ATP substrates Cg Co-crystallization experiments were performed by adding to MetK crystals, and the complex structure was successfully confirmed at a resolution of 2.4 Å (Table 1). Cg The crystal structure of the MetK complex belonged to the P21 space group, and four molecules were present in the asymmetric unit. Interestingly, however, despite the addition of methionine and ATP substrates, the inventors observed electron density maps for adenosine and triphosphate at the active site (Fig. 3A). The inventors hypothesized that a reaction occurs during the co-crystallization experiment, and that intermediates and products bind to the active site. Furthermore, the inventors [inferred that] Mg at the active site 2 + and K + An electron density map for metal ions was observed (Fig. 3A).

[0077] The adenosine molecule was stabilized by residues of Asp178, Gly248, Phe250, and Asp258. The purine ring of adenosine formed pi-pi interactions with Phe250, and the ribose group formed hydrogen bonds with the side chains of Asp178 and Asp258. Additionally, the amino group of the purine ring interacted with the main chain of Gly248 (Fig. 3B). K + The ion bound to the side chains of Glu60 and Asp258 (Fig. 3B). One Mg 2 + The ion binds to the side chain of Asp21, and the remaining Mg 2 + The ions bound to the side chains of Asp133 and Asp291 (Fig. 3C). Two Mg 2 + The ion was significantly involved in the binding of the triphosphate molecule (Fig. 3C), which was also stabilized via hydrogen bonding by the side chains of His19, Asp21, Lys180, Arg264, Lys265, and Lys285 and the main chain of Ala281 (Fig. 3C). The catalytic His19 residue was located between the adenosine and triphosphate molecules (Fig. 3C). CgWhen comparing the amino acids forming the active site of MetK with other MetKs, most of the amino acids were found to be significantly conserved with MetK enzymes (Fig. 1B), which indicates that the enzymes share major residues for the acceptance of substrates and metal ions.

[0078] < Examples 4> By SAM Cg MetK's Allosteric control

[0079] Interestingly, even though SAM was not added during the co-crystallization process, the inventors observed a strong density map for SAM at an unexpected site (Fig. 4A). The inventors hypothesized that SAM products are formed during the co-crystallization experiment involving the addition of methionine and ATP substrates. One SAM molecule binds to each monomer. SAM binding sites are formed on the tetramerization surface, Cg Two SAM molecules binding to MetK were located close to each other (Fig. 4A). The SAM molecules are stabilized through hydrogen bonding between the main chains of Val61, Thr63, Val67, and Gly101 and the side chains of Tyr66 and Glu68 (Fig. 4B).

[0080] More interestingly, the SAM-binding site was located near the active site, which supports the idea that the binding of SAM influenced the shape of the active site. To confirm the effect of SAM binding on the active site, the inventors Cg The structure of the MetK complex is a complex that uses methionine as a substrate. Ec It was compared with the structure of MetK. The region composed of Gln103-Glu125 lacks methionine. Cg In the MetK structure, it is disordered, whereas in the methionine-complex structure EcIn MetK, a helix is ​​formed in the corresponding region, significantly contributing to the binding of methionine substrates (Fig. 4C). As previously mentioned, Gly101 is Cg It is involved in the binding of SAM in MetK (Fig. 4B), and the inventors hypothesized that the interaction between Gly101 and SAM interferes with the proper formation of the methionine-binding domain and, consequently, may interfere with substrate binding (Fig. 4C). The structural observation is Cg This supports the idea that MetK can be interfered with by SAM products that use allosteric inhibitor binding sites for SAM located near the active site.

[0081] The inventors of the present invention Cg Enzyme kinetics experiments of MetK were performed, K m and k cat The values ​​for ATP are 0.618 mM and 0.544 s, respectively. -1 For methionine, 0.1 mM and 0.471 s, respectively -1 It was shown as (Figs. 4D and 4E). Subsequently, the inventors performed inhibition kinetics experiments to investigate the inhibition mode of the enzyme by adding SAM products at various concentrations. As a result of the experiment, as the SAM concentration increased, K m Increase in value and V max It showed a decrease in values, which is not a typical inhibition pattern (Fig. 4E and Table 2). Based on the above results, the inventors hypothesized that inhibition was induced by SAM products binding not only to the active site but also to the allosteric site, which is due to SAM Cg This indicates that MetK inhibition occurs both competitively and non-competitively. Therefore, the results of the inhibition kinetics were applied to a mixed inhibition equation, Cg The Ki and alpha (α) values ​​of MetK were 0.0241 mM and 12.7, respectively (Fig. 4E and Table 2).

[0082] Cg MetK WT Cg MetK E68A SAM (mM) Vmax(mM / min) Km (mM) Ki (mM) Alpha (α) SAM (mM) Vmax(mM / min) Km (mM) Ki (mM) Alpha (α) 0 28.28 0.1 0.024 12.8 0 31.36 0.116 0.878 1.36 0.05 24.32 0.264 0.1 28.94 0.119 0.1 21.34 0.388 0.2 26.87 0.122 0.15 19.00 0.485 0.5 22.13 0.129

[0083] The equation used to analyze the inhibition constant (Ki, α) is ' v = (Vmax*[S]) / [ K m * (1 + [I] / Ki)+S* (1+[I] / α*Ki)]'. [S] and [I] are the substrate concentration and inhibition concentration, respectively. The R-squared value of the appropriate inhibition model was 0.98 or higher.

[0084] < Examples 5> By rational protein engineering Cg MetK's Reduction in product inhibition

[0085] Since SAM is used in various biosynthetic pathways, reducing enzyme inhibition by SAM can be a better strategy to improve SAM utilization pathways. Accordingly, the inventors [developed] a structure-guided protein engineering [developed] a [developed] SAM inhibition reduced Cg Attempts were made to develop MetK variants. As previously mentioned, the side chains of Tyr66 and Glu68 within the allosteric site interact with SAM through hydrogen bonding (Fig. 4B). The inventors substituted these residues with alanine, which is expected to reduce the binding affinity of SAM at the allosteric binding site. In the absence of SAM Cg MetK Y66A and Cg MetK E68A When measuring the related activity of the variant, Cg MetK Y66A The variant showed reduced activity, and Cg MetK E68A The variant Cg MetK WT It exhibited slightly higher activity (Fig. 5A). Surprisingly, in the presence of 0.1 mM SAM, the activity of the variant was nearly similar to that in the absence of SAM, indicating significant inhibition of release by the variant's SAM product. In particular, Cg MetK E68A Compared to the case without SAM addition, the variant maintained approximately 95% activity upon the addition of 0.1 mM SAM (Fig. 5A). Furthermore, in the presence of 0.1 mM SAM Cg MetK E68A The activity of the variant is in the absence of SAM. Cg MetK WT It was higher than (Fig. 5A). Cg MetK E68A As a result of the inhibition kinetics experiments of the variant, the Ki level increased significantly to 0.878 mM, while the α level decreased significantly to 1.36 (Fig. 5B and Table 2). In addition, the E68A mutant Cg The thermal stability of MetK was also increased by 15°C (Fig. 5C).

[0086] To explain the structural basis for inhibition reduction, Cg MetK E68A The crystal structure of the variant was confirmed at 1.8 Å (Table 1). Cg MetK E68A In the structure, the Tyr66 residue is rotated nearly 180° and forms a new hydrogen bond with the Glu102 residue, which causes a disruption in the conformation of the SAM allosteric binding site (Figs. 5D and 5E). Another monomer provides the Tyr66 and Glu102 residues, and the formation of hydrogen bonds between these two residues can enhance tetramerization, which Cg MetK E68A Explains the increase in thermal stability of the variant (Fig. 5F).

[0087] In summary, the inventors Cg The structures of the apo and substrate-complex forms of MetK were confirmed. Based on the complex structure, Mg for the enzyme2 + and K + The binding mode between metal ions and substrates was identified. In addition, the inventors... Cg It was shown that MetK possesses an allosteric inhibitory binding site for SAM products near its active site, and inhibition kinetics experiments supported that the enzyme is inhibited competitively and non-competitively by SAM. In addition, the inventors performed rational protein engineering to reduce enzyme inhibition for SAM products, dramatically reducing inhibition by SAM, and Cg MetK WT Exhibiting improved activity and thermal stability compared to Cg MetK E68A A variant was developed. This invention demonstrated how structural information can be used to reduce enzyme inhibition by enzyme products and how it can be applied to improve various SAM-utilizing cellular pathways.

[0088] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 delete Claim 2 delete Claim 3 An amino acid sequence in which the 68th amino acid, glutamic acid (E), in the amino acid sequence represented by SEQ ID NO. 1 is substituted with alanine (A), with reduced inhibition of enzyme activity by S-adenosylmethionine (SAM) and increased thermal stability Cg MetK variant. Claim 4 Paragraph 3 Cg Polynucleotide encoding a MetK variant. Claim 5 A recombinant vector comprising the polynucleotide of claim 4. Claim 6 Microorganism transformed with the recombinant vector of paragraph 5. Claim 7 A step of culturing the microorganism of claim 6; and from the cultured microorganism, the of claim 3 Cg A method comprising the step of obtaining a MetK variant, wherein enzyme activity inhibition by SAM is reduced and thermal stability is increased Cg Method for producing MetK variants. Claim 8 A space group of C121, unit cell parameters of a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, α = γ = 90°, β = 128.91°, and composed of an amino acid sequence represented by SEQ ID NO. 2, Cg Crystal of the MetK variant. Claim 9 i) a reservoir solution comprising 10 to 50% (v / v) PEG 20,000 and 0.01 to 1 M MES with a pH of 6.0 to 8.0, and ii) an amino acid sequence represented by SEQ ID NO. 2 Cg A crystalline form having the following, comprising the step of mixing protein solutions containing MetK variant proteins together and crystallizing them by a vapor diffusion method. Cg Crystallization method of MetK variant: [Crystal morphology] The space group is C121, the unit cell parameters are a = 150.4 Å, b = 125.44 Å, c = 116.31 Å, and α = γ = 90°, β = 128.91°. Claim 10 i) Paragraph 8 Cg Using the crystalline stereostructure of the MetK variant Cg MetK activity regulating candidate peptides or Cg A step of generating or screening MetK binding candidate compounds; and ii) the candidate peptide or compound generated or screened in step i) Cg A step including determining whether it regulates MetK activity, Cg Screening method for MetK activity modifiers.

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