Stereostructure of decarbamylase and uses therof

专利摘要:
An object of the present invention is to clearly identify the steric structure by X-ray crystal structure analysis of decarbamylase, and to improve the reactivity to DN-carbamoyl-α-amino acids, which are substrates, by using the steric structure. Molecular design to provide a decarbamylase mutant excellent for industrial use. Specifically, the present invention relates to a three-dimensional model of a decarbamylase determined by X-ray crystal structure analysis, a three-dimensional model of a mutant and a complex three-dimensional model of a substrate, a product and the like, and a molecular design method using the three-dimensional structure. The present invention relates to a method for obtaining a decarbamylase mutant using the method, and a method for designing and preparing a decarbamylase mutant obtained by the method and a protein mutant having a structure similar to the decarbamylase.
公开号:KR20020037044A
申请号:KR1020027002725
申请日:2000-08-30
公开日:2002-05-17
发明作者:나카이다카히사；모리카와소이치；이시이기요토；난바히로카즈；야지마가즈요시；이케나카야스히로；다카하시사토미
申请人:다케다 마사토시；가네가후치 가가쿠 고교 가부시키가이샤；
IPC主号:

专利说明:

STEREOSTRUCTURE OF DECARBAMYLASE AND USES THEROF}
[2] Optically active D-α-amino acids are important compounds as pharmaceutical intermediates, and in particular, D-α-amino acids, intermediates for the preparation of semi-synthetic penicillins or semisynthetic cephalosporins. D-phenylglycine, D-para-hydroxyphenlyglycine and the like are known as examples of industrially useful compounds. As a manufacturing method of such D- (alpha)-amino acids, the method of removing the carbamoyl group of corresponding DN-carbamoyl amino acids and obtaining them is already known as a well-known technique, At this time, the carbamoyl group is removed by a chemical method or a method using an enzymatic reaction of a microorganism.
[3] The enzyme that removes the carbamoyl group is called decarbamylase. This enzyme catalyzes the conversion of DN-carbamoyl amino acids to D-α-amino acids. This enzyme is of the genus Pseudomonas, Genus Agrobacterium, Genus Aerobacter, Genus Aeromonas, Genus Brevibacterium, Genus Bacillus, Flavo bacteria (Flavobacterium), Settatia, Micrococcus, Arthrobacter, Algenogenes, Achromobacter, Morocco It has been identified in the genus Moraxella, in the genus Pararacoccus, in the blastbacter and in the comamonas. The amino acid sequence and / or nucleic acid sequence of decarbamylase has been determined in the genus Agrobacteria, for example Agrobacterium radiobacter NRRL B11291 and Agrobacterium sp. ( Agrobacterium sp.KNK712 (Kaneka bacteria)) is mentioned.
[4] In general, enzymes do not have sufficient stability to withstand the conditions of industrial use, which are used for reaction at room temperature or high temperature, so the stability affects the cost of the product. There are many cases. In addition, as a means for facilitating an enzymatic reaction, it is repeatedly used as a so-called "bioreactor" such as an immobilized enzyme or an immobilized bacterium. In this case, the use of the enzyme is limited by the stability of the enzyme, which greatly affects the cost of the product.
[5] In order to effectively use an enzyme that catalyzes a useful reaction in industry, a method for quickly and efficiently making an enzyme having excellent physical properties such as stability or catalytic ability is desired. As a method of obtaining an enzyme having improved physical properties or function, an artificial mutation is added to a gene encoding an enzyme by chemical treatment or enzyme treatment, and a recombinant DNA containing the mutation is added. So-called random screening methods are widely known for screening enzymes that enter a host cell and have desired functions and / or properties. On the other hand, according to the recent advances in structural biology, three-dimensional structures of proteins such as enzymes are clearly identified by X-ray crystallography or NMR analysis, and modification or physical properties of proteins using the three-dimensional structure and molecular design method (() ) Is also very active.
[6] Here, the "protein conformation" refers to a three-dimensional structure of a protein that is folded under the conditions of a protein having an amino acid sequence, that is, defined by a certain condition and the amino acid sequence. The steric structure of a protein is determined by X-ray crystallography (X-ray crystallography) or nuclear magnetic resonance, for example.
[7] Regarding the modification of the properties of decarbamylase, we have succeeded in screening enzyme-producing bacterial strain (E. coli JM109) which improved heat resistance by random screening method. Advantageous decarbamylase mutants have been obtained (International Publication No. WO94 / 03613 Pamphlet). In addition, a mutant (Japanese Patent Laid-Open No. 9-173068) with improved stability was obtained by site-specific mutagenesis. However, these examples produce mutants based only on the primary sequence of amino acids and do not utilize rational molecular design using the conformation of enzymes.
[8] Until now, as decarbamylase and its similar enzyme (그), it is not known that the three-dimensional structure is clear. Decarbamylase is an amidase in the interpretation of sequence similarity to PIR Release57 (NCBI), a database of protein sequences with known amino acid sequences. It has been found that about 25-30% of hydrolase, such as nitrilease and nitrilease, has weak sequence similarity. However, none of the conformations of these enzymes with weak sequence similarity have been clarified so far. For this reason, the so-called homology modeling technique (e.g. Swiss-Pdbviewer (modeling program) (Swiss Institute of Bioinformatics (SIB), ExPASy Molecular), which uses the conformation of pseudoproteins often used in molecular design methods. Biology Server (available at http://www.expasy.ch/); Guex, N. and Peitsch, MC (1997) SWISS-MODEL and the Swiss-Pdbviewer: An environment for comparative protein modeling, Electrophoresis 18 , 2714-2723), it was virtually impossible to estimate the conformation of decarbamylase. For example, it is difficult to obtain a conformational model with sufficient precision in order to apply a reasonable molecular design method due to the weak sequence similarity, which is less than 30% even if the conformation of an analogous enzyme is determined. Precise Atomic Coordinate Data of Decarbamilase To Predict Highly Accurate Amino Acid Mutation Related to Changes in pH and Inactivation of Enzymatic Reactions ) Is essential. Determination of the three-dimensional structure of decarbamylase makes it possible to analyze the precise three-dimensional structure and to apply a reasonable molecular design method based on it. Furthermore, it is possible to promptly modify modified enzyme (改變 酵素) which is advantageous for industrial use. It is also possible to obtain efficiently.
[1] The present invention is an enzyme for converting DN-carbamoyl amino acid (D-α-amino acid) to the corresponding D-α-amino acid (hereinafter referred to as decarbamylase) (decarbamylase) of the crystal (結晶: crystal). In addition, the present invention relates to a stereostructure of decarbamylase determined by analysis of X-ray crystallography using the crystal and its use, in particular its Stability of decarbamylase using three-dimensional structure, resistance to organic solvents, resistance to air oxidation, etc., optimum conditions for enzyme reactions, pH change and inactivity: The present invention relates to a method for designing a mutation of an amino acid related to specific activity improvement. The present invention also relates to a method for producing a decarbamylase mutant using the three-dimensional structure, the obtained decarbamylase mutant and its use.
[15] 1 is a topology showing the protein conformation of decarbamylase. The β strand structure is triangular, and the α helix structure is a circle. "N-term" represents the end of the network, and "C-term" represents the carboxy end.
[16] FIG. 2 is a diagram showing a ribbon-strand diagram of decarbamylase. FIG. The β-stranded structure is shown as a plate, and the α-helical structure is shown as a helix. The side chains of amino acids involved in catalytic activity are represented by balls and sticks.
[17] Figure 3 is a schematic diagram showing the matrix binding mode of the active site (active site: active site) of decarbamylase. R is the side chain of D-α-amino acid. The residue number (residue number: 殘 基 番號) and the name of the amino acid in three letter notation.
[18] 4 is a view showing a three-dimensional structure of the catalytically active site of decarbamylase. The β-stranded structure is shown in a plate shape, the α helix structure is represented by a helix, and the side chains of amino acids involved in catalytic activity are represented by spheres and sticks. Amino acid names are indicated by residue number and three letter designation.
[19] FIG. 5 shows a Harker plane of a difference Patterson map calculated from diffraction data of 1.00 Hz and 0.98 Hz. FIG.
[20] Fig. 6 is a diagram showing a Hacker plane of difference Patterson's degree calculated from diffraction data of 1.00 Hz and 1.27 Hz.
[21] Fig. 7 is a diagram showing the Hacker plane of the difference patternson diagram calculated with 0.98 Hz as anamalous data.
[22] Fig. 8 is a representative electron density map of the catalytically active site showing the three-dimensional structure in the present invention.
[9] In order to solve this problem, an object of the present invention is to obtain a single crystal of decarbamylase, which is currently provided for industrial use, and to clearly identify its three-dimensional structure by X-ray crystal structure analysis. do. In addition, the present invention is a molecule aiming to improve the reactivity to the substrate DN-carbamoyl-α-amino acids using the steric structure of decarbamylase, optimization of the reaction pH, stability of heat and air oxidation, etc. It is an object of the present invention to provide a decarbamylase mutant excellent in industrial use by designing and to provide a method for producing D-α-amino acid using the obtained decarbamylase mutant.
[10] MEANS TO SOLVE THE PROBLEM As a result of earnestly examining in order to solve the said subject, the present inventors obtained the single crystal | crystallization of decarbamylase and the heavy atom derivative crystal (heavy atom derivative crystal), and the heavy atom isomorphism of these crystals. Decarbamylase was analyzed by X-ray crystal structure analysis using the heavy atom isomorphous replacement method and the multi-wavelength anomalous dispersion method. The present invention has been completed by determining precise conformations and making mutants with improved properties based on these conformations.
[11] The present invention relates to a space group P2 ₁ 2 ₁ of an orthorhombic system or an amino acid sequence represented by SEQ ID NO: 1 or a space group of a tetragonal system P2 ₁ 2 ₁ A decarbamylase crystal having an amino acid sequence represented by 2 ₁ and SEQ ID NO: 2 is disclosed. Decarbamylase having a crystal having an amino acid sequence represented by space group P2 ₁ 2 ₁ 2 and SEQ ID NO: 1 or an amino acid sequence represented by space group P2 ₁ 2 ₁ 2 ₁ and SEQ ID NO: 2 It's about the decision. In one embodiment, the crystal has a unit cell having a rectangular parallelepiped shape, and has a lattice constant: a = 66.5 to 68.5 kW, b = 135.5-138.0 ms, c = 66.5-68.5 Hz. The amino acid sequence is SEQ ID NO: 1. In another embodiment, the crystal has a unit grid having a rectangular parallelepiped shape, and a unit lattice constant: a = 68.5-70.5 Å, b = 138.0-140.5 Å, c = 68.5-73.0 Å. In another embodiment, the crystal has a unit lattice having a rectangular parallelepiped shape, and a unit lattice constant: a = 81.5-82.5 ms, b = 133.0-135.0 ms, c = 119.5-121.5 ms. The amino acid sequence is SEQ ID NO: 2. In one aspect, the invention can provide a crystal comprising at least one heavy metal atom per molecule of decarbamylase in the crystal. In one embodiment, the heavy metal atom is any one of mercury, gold, platinum, lead, iridium, osmium and uranium. In another embodiment, the present invention can provide frozen crystals prepared by freezing decarbamylase crystals in a liquid nitrogen state.
[12] In another aspect, the present invention provides a method for producing decarbamylase crystals, the step of supplying a decarbamylase solution at a concentration of 1 to 50 mg / ml, polyethylene glycol (PEG) at a concentration of 5 to 30% by weight : A process for supplying a precipitant solution containing polyethylene glycol) or methoxypolyethylene glycol (PEGMME) and a buffer having a pH concentration of 6.0 to 9.0, wherein the decarbamylase solution And a step of mixing the precipitated solution with the precipitant solution and allowing the obtained mixed solution to stand for a predetermined period until the decarbamylase crystals in the solution grow to a predetermined size or more. to provide. In one embodiment, the method of the present invention further comprises the step of the mixing comprising mixing the droplets of the decarbamylase solution with the droplets of the precipitant solution. The process includes suspending the mixed droplets obtained in the mixing process in a sealed reservoir containing a precipitant solution in a closed container, wherein the vapor pressure of the precipitant solution in the solution tank Lower than vapor pressure. The method of the present invention further includes the step of mixing the droplets of the decarbamylase solution with the droplets of the precipitant solution, and the step of allowing the stationary process to mix the mixed droplets obtained in the mixing step in an airtight container. Suspending in the droplet zone of the solution bath containing the precipitant solution, wherein the vapor pressure of the precipitant solution in the solution bath is lower than the vapor pressure of the mixed droplets. In another embodiment, the time period for allowing the mixed solution in the method of the present invention to stand is from 1 day to 3 weeks. In another embodiment, the method further comprises disposing the decarbamylase solution in a size exclusion semi-permeable membrane after the step of supplying the solution of decarbamylase. And the step of mixing includes diffusing the precipitant solution into the decarbamylase solution through the semipermeable membrane. In another embodiment, the mixing step in the method of the present invention includes gradually adding the precipitant solution to the decarbamylase solution, wherein the step of leaving the mixture is allowed to stand in the sealed container. It includes. In another aspect, the present invention relates to a decarbamylase characterized by a steric structure having the steric methoxy polyethyl topology of the protein shown in FIG. In one embodiment, the present invention is characterized by having a four-layer sandwich structure comprising a secondary structure comprising four α helix and 12 β sheets. It relates to decarbamylase. In a preferred embodiment of the present invention, the amino acid residue involved in the enzymatic reaction is one residue of cystein, two residues of glutamic acid and one residue of lysine, and the substrate of the enzyme reaction. This DN-carbamoyl-α-amino acid (DN-carbamoyl-α-amino acid) is characterized by the steric structure of the active site having the substrate-binding mode shown in FIG. In another embodiment of the invention, the invention is an enzyme molecule having decarbamylase activity, wherein at least the following amino acids in SEQ ID NO: 1 or 2 are: Glu at position 46, Lys at position 126, position 145 The present invention relates to an enzyme molecule having an active site cavity formed from Glu and amino acids corresponding to Cys at position 171. In another embodiment, in the active site cavity, the DN-carbamoyl-α-amino acid reacts with Lys at position 126, His at position 143, and Glu at position 145 at the time of reaction. And an amino acid corresponding to Arg at position 174, Arg at position 175, and Thr at position 197. In another embodiment, in the active site cavity, amino acids corresponding to Glu at position 46, Glu at position 145, and Cys at position 171 in SEQ ID NO: 1 or 2 are hydrogen bonded via water molecules. . In another embodiment, the DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine, DN-carbamoyl-parahydroxyphenylglycine (DN-carbamoyl -parahydroxyphenylglycine), DN-carbamoyl-phenylalanine, DN-carbamoyl-valine, DN-carbamoyl-alanine, DN DN-carbamoyl-cysteine, DN-carbamoyl-asparatic acid, DN-carbamoyl-glutamic acid, DN-carbamoyl-glutamic acid DN-carbamoyl-glycine, DN-carbamoyl-histidine, DN-carbamoyl-isoleucine, DN-carbamoyl-isoleucine (DN-carbamoyl-isoleucine) DN-carbamoyl-lysine, DN-carbamoyl-leucine, DN-carbamoyl-methionine, DN-carbamoyl-methionine DN-carbamoyl-asparagine, DN-carbamoyl-proline, DN-carbamoyl-glutamine, DN-carbamoyl-arginine -arginine, DN-carbamoyl-serine, DN-carbamoyl-threonine, DN-carbamoyl-tryptophan and DN -Carbamoyl-tyrosine (DN-carbamoyl-tyrosine). In another embodiment, the present invention relates to a decarbamylase or a mutant thereof and a DN-carbamoyl-α-amino acid, which is constructed by a molecular design method from the steric structure of decarbamylase in the present invention. Or to a decarbamylase complex characterized by a complex conformation with D-α-amino acids.
[13] In one aspect of the present invention, the present invention provides a method for designing a decarbamylase mutant, wherein the three-dimensional structure of the decarbamylase according to any one of claims 14, 16, and 21 is used. The present invention relates to a method comprising the step of designing a decarbamylase mutant that modifies physical properties and / or functions based on structure. In another aspect, the present invention provides a method for designing a decarbamylase mutant, comprising: a step of generating a crystal of an enzyme having decarbamylase activity and an X-ray crystal structure analysis of the crystal (X-ray crystallography: a process of determining the steric structure of the crystal by X-line analysis and designing a decarbamylase mutant having improved physical properties and / or functions based on the determined steric structure It relates to a method for designing a decarbamylase mutant. In another aspect, the present invention is a method for producing a decarbamylase mutant, the method comprising the steps of producing a crystal of the enzyme having decarbamylase activity, X-ray crystal structure of the crystal A process of determining the steric structure of the crystal by analysis, a process of designing a decarbamylase mutant having improved physical properties and / or function based on the determined steric structure, and a process of producing the decarbamylase mutant It is about a method of including. In one embodiment, the three-dimensional structure used by the method of this invention is a three-dimensional structure of decarbamylase in this invention. In another embodiment, the step of designing the decarbamylase mutant may include the step of designing the decarbamylase mutant, such as alteration of substrate specificity of enzyme, alteration of enzyme inactivation, The purpose of the present invention is to modify one or more enzyme properties selected from the group consisting of improving the stability of the enzyme, optimizing the optimum pH of the enzyme, and changing the water solubility of the enzyme. In another embodiment, the method for producing the decarbamylase mutant aims at improving the stability of the enzyme. Preferably, the design of the mutant for improving the stability of the enzyme comprises a mutation that replaces an amino acid residue that results in a decrease in activity by air oxidation. In another embodiment, altering the enzyme properties includes altering enzyme inactivity and optimizing the optimum pH.
[14] In another aspect, the present invention relates to a decarbamylase mutant obtained by the method of the present invention. The present invention also provides a method for screening and / or designing an inhibitor of decarbamylase using the steric structure in the present invention. In another aspect, the present invention provides the use of a three-dimensional structure of decarbamylase crystals or a three-dimensional structure of decarbamylase to show that at least 30% similarity of the amino acid primary sequence to decarbamylase. Provided is a method for modifying a separate polypeptide (peptide) enzyme or protein enzyme comprising.
[23] EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.
[24] As used herein, the term "decarbamylase activity" refers to a carbamoyl group that modifies the amino acid of DN-carbamoyl-α-amino acid. It refers to the activity of removing the group) and converting it to D-α-amino acid. "Decarbamylase" refers to an enzyme having decarbamylase activity. Examples of decarbamylase include enzymes having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Decarbamylase having the amino acid sequence of SEQ ID NO: 1 is agrobacteria sp. It is isolated from KNK712 ( Agrobacterium sp. KNK712) to determine the arrangement. An enzyme having the amino acid sequence of SEQ ID NO: 2 is an enzyme obtained from E. coli mutants (E. coli strains) by screening by random mutations in SEQ ID NO: 1.
[25] As used herein, the term "mutant" of decarbamylase or other enzyme mutant means that at least one amino acid in the amino acid sequence of the original enzyme is substituted, added, or deleted. refers to a modified enzyme having an amino acid sequence modified or modified to receive at least a portion of the activity of the original enzyme. An "active fragment" refers to a fragment having a part of an amino acid sequence of a certain protein enzyme or a polypeptide enzyme, and a fragment containing at least a part of the activity of the original enzyme. By "at least part of the activity" is typically meant at least 10% specific activity of the original enzyme, preferably at least 50% of the original enzyme, but less than 10% inactivity if desired. Sometimes you say.
[26] As used herein, the term "native crystal" refers to decarbamylase as a certain precipitating agent such as ammonium sulfate, polyethylene glycol, or added salts. It refers to a crystal that grows up to a single crystal in a buffer containing an appropriate composition and does not contain heavy metal atoms.
[27] As used herein, the term "heavy atom derivative crystal" refers to any one of the following crystals: (i) The prepared natural crystals are mercury, gold, platinum, lead, Heavy metal atoms are added to decarbamylase in the crystal without breaking crystallinity by immersion in a solution containing heavy metal compounds such as iridium, osmium and uranium. Crystals bound by covalent or coordination bonds, or (ii) decarbamila in a solution containing any of the precipitants, additives such as polyethylene glycol, polyethylene glycol, etc., in an appropriate composition, and containing the heavy metal compound at an appropriate concentration. Crystals wherein the agent is grown to single crystals and (iii) Methionine and / or succinate of decarbamylase It refers to a crystal obtained by using a mutant in which a cystein residue is substituted with selenomethionine and / or selenocysteine.
[28] In one embodiment of the present invention, the crystal of decarbamylase having the amino acid sequence of SEQ ID NO: 1 or 2, the concentration and pH of the solution to be used within the prescribed range (regarding the concentration of decarbamylase 1 to 50 mg / ml, polyethylene glycol (PEG) with careful control to 5-30% by weight with respect to the concentration of polyethylene glycol or methoxypolyethylene glycol, 6.0-9.0 for pH Or by growing in a precipitant solution containing methoxypolyethylene glycol (PEGMME), buffer and optional addition salts. One of the three basic techniques commonly used for crystal growth of proteins: vapor diffusion method, dialysis method, and batch method (Methods in Enzymology) , Vol. 114, Diffraction Methods for Biological Macromolecules Part A or Vol. 276 Macromolecular Crystallography Part A), although vapor diffusion is preferred.
[29] Steam diffusion involves placing droplets of a protein solution containing precipitant in a container containing a buffer solution containing a higher concentration of precipitant (external solution), then sealing and standing. ) There is a hanging-drop method and a seating-drop method according to the method of dropping. The conventional method places a droplet of protein solution on a cover glass and seals the cover glass by inverting it on a reservoir. On the other hand, in the dropping drop method, an appropriate droplet stage (液滴 臺) is installed inside the solution tank, and droplets of the protein solution are disposed on the droplet bed to seal the solution tank with a cover glass or the like. The solution in the solution bath contains a precipitant, which is present in small amounts in the droplets of protein. The precipitant solution used in the vapor diffusion method is formed to contain the following components: (a) PEG or PEGMME having a molecular weight of 4000 to 9000, preferably an average molecular weight of 7500 and a concentration of 10 to 20% by weight, (b) addition Salt, sodium chloride, magnesium chloride having a concentration of 0.1 to 0.5 M as a salt (best results are obtained with 0.2 M lithium chloride) and (c) pH 6.5 to 8.0, preferably pH 7.5 Sufficient amount of buffer. HEPES (Sigma, St Louis, MO, USA) of 0.05 to 0.1 M can be used for this purpose. Other buffers such as sodium phosphate, calcium phosphate and tris (hydroxymethyl) aminomethane malate are also used.
[30] The "batch method" refers to the addition of a precipitant solution to a protein solution little by little, while centrifuging and removing insoluble matters in a slightly clouded state. (密封) After the method of politics. In addition, "dialysis method" refers to a method of dialysis of a protein solution using a semipermeable membrane in a buffer (external solution) containing a precipitant (Methods in Enzymology, Vol. 114, Diffraction Methods for Biological Macromolecules Part A).
[31] In the present specification, the "predetermined size" means the minimum size that can be measured by X-ray crystallography (X-ray crystallography), and in the case of the decarbamylase of the present invention, preferably 0.3 X 0.3 x 0.1 mm. In addition, the "predetermined period" refers to a sufficient period of time required for the size of the crystal to reach a predetermined size or more, and in the case of the decarbamylase of the present invention, preferably 1 day to 3 weeks.
[32] The natural crystal of decarbamilase of SEQ ID NO: 1 having a predetermined size suitable for the analysis of the prepared X-ray crystal structure as described above has (1) a diamond-shaped plank shape, and ( 2) Crystals with the same appearance have different lattice constants (單位 格子 定 數). In addition, although not suitable for X-ray crystal structure analysis, by selecting appropriate conditions such as precipitants, buffers, etc., small or small crystals having a needle-like or columnar shape can be obtained. Regarding the use of such crystals, enzymatic reactions can be stabilized for a long time even in the presence of an organic solvent by crosslinking microcrystals of enzymes with protein crosslinking reagents such as glutaraldehyde. A technique called cross-linked enzyme crystal (CLEC) has been reported (NL St. Clair & MA Navia, (1992) J. Am. Chem. Soc. 114, 7314-7316). The crystal of decarbamylase obtained by the present invention expands the scope of application of the CLEC technique.
[33] In one embodiment of the present invention, a heavy atom derivative crystal effective for X-ray crystal structure analysis, that is, a crystal in which heavy metal atoms are bonded to a protein in the crystal while accommodating crystallinity of a natural crystal is provided. The determination of heavy atom derivatives is based on the heavy atom isomorphous replacement method and the multi-wavelength anomalous dispersion method, which are the basic techniques of X-ray crystal structure analysis of proteins. It is used when applying the method. The heavy atom derivative crystal of decarbamylase is a soaking method in which a crystal is immersed in a solution containing a heavy metal compound which does not dissolve or disintegrate and stably preserves at least several days and supplies a desired concentration. It can be prepared by. Heavy metal compounds used in the immersion method are metal salts or organometallic compounds including gold, platinum, iridium, osmium, mercury, lead, uranium, samarium and the like. In the heavy metal soaking method, a heavy metal compound at a concentration of 0.1 to 100 mM, for example, mercury compound EMTS (ethyl mercury thiosalicylate sodium), calcium dicyanogold (I) A preservative solution containing a suitable precipitant and addition salt composition containing (patassium dicyanoaurate (I)) and the like to supply a desired pH is used. A preferred example of a preservation solution is 0.01 M HEPES buffer (pH 7.5) containing 20-30% by weight polyethylene glycol 6000 and 0.2 M lithium chloride. Determination of whether heavy metal atoms are introduced into the crystal, ie bound to the protein in the crystal, has already been made by collecting the X-ray diffraction intensity data of the crystal prepared by the immersion method. The diffraction intensity data of the natural crystals present are compared.
[34] Or microorganisms that produce decarbamylase (e.g., recombinant E. coli containing DNA of the genus Agrobacterium) and contain selen, a heavy metal atom. The methionine or cysteine residue of decarbamylase is transformed into selenomethionine or selenocysteine by growing and culturing in a medium containing selenomethionine or selenocysteine. Substituted mutants are obtained. In this way, when decarbamylase in which heavy metal atoms are introduced into the protein without using the immersion method is obtained, the heavy atom derivative crystal can be prepared by crystallization using the above conditions and the like.
[35] In general, it is known that crystals of proteins are significantly damaged by X-rays, so it is important to obtain crystals that are difficult to be damaged in order to succeed in analyzing the X-ray crystal structure. Recently, attempts have been made to obtain high quality and high resolution diffraction data by freezing crystals and measuring diffraction data in the frozen state (Methods in ENZYMOLOGY Vol. 276, Macromolecular Crystallography). , Part A, CW Carter, Jr and RM Sweet, [13] Practical Cryocrystallography (DWRodgers)). In general, freezing of protein crystals has been devised, such as treatment with a solution containing a freezing stabilizer such as glycerol in order to prevent the collapse of the crystals by freezing. In the present invention, freezing crystals of natural crystals and heavy atom derivatives of decarbamylase can be prepared by removing crystals in crystallization droplets or immersion solutions without adding a freeze stabilizer and directly immersing them in liquid nitrogen and freezing them instantaneously. . Freezing crystals can also be prepared by performing the above-mentioned operation of temporarily freezing the crystal contained in a preservation liquid containing a freeze stabilizer.
[36] The three-dimensional structure of a similar protein has not yet been identified, and new proteins, such as decarbamylase, which cannot be analyzed by the molecular replacement method using the three-dimensional structure. In the determination of conformation, Methods in ENZYMOLOGY Vol. 115, Diffractipn Methods for Biological Macromolecules, Part B, HWWyckoff, CHW Hirs and SN Timasheff, and Methods in ENZYMOLOGY Vol. 276, Macromolecular Crystallography, Part A, CW Carter, Jr. and RM Sweet) or multi-wavelength anomaly dispersion (SN Timasheff and Methods in ENZYMOLOGY, Vol. 276, Macromolecular Crystallography, Part A, CW Carter, Jr. and RM Sweet). That is, to calculate the electron density from the difference in diffraction intensity between the diffraction data of the natural crystal and the heavy atom derivative crystal or the diffraction intensity difference between the diffraction data measured at different wavelengths. The three-dimensional structure can be determined by obtaining the initial phase. In three-dimensional structure determination of decarbamylase to which the heavy atom isomorphic substitution method or the multi-wavelength abnormal dispersion method is applied, as a heavy metal atom, for example, mercury, gold, platinum, uranium, selenium atom, etc. are contained. It is obtained using a heavy atom derivative crystal. Preferably, the heavy atom derivative crystal obtained by the immersion method using the mercury compound EMTS or calcium dicyano gold (I) is used.
[37] The diffraction data of the natural crystals and the heavy atom derivative crystals can be obtained from the beam line for protein crystal structure analysis of R-AXIS IIc (rigaku Electric) or SPring-8 (Nishiharima large emission facility). Can be measured using. Diffraction data measurement at multiple wavelengths for applying the multi-wavelength anomalous dispersion method can be carried out using the SPring-8 beamline for protein crystal structure analysis. The measured diffraction image data is processed into reflection intensity data using a data processing program or a program DENZO (McScience) or the same image processing program (or single crystal analysis software) attached to the R-AXIS IIc. do. In the reflection intensity data of the natural crystals and the reflection intensity data of the heavy atom derivative crystals at multiple wavelengths, the position of the heavy metal atoms bound to the protein in the crystal is determined by using a difference Patterson map. After retrieval, the initial phase can be refined by using the program PHASES (W. Furey, University of Pennsylvania or CCP4 (British Biotechnology & Biological Science Research Counsil, SERC) or by using the same diffraction data analysis program to refine the heavy atom position parameter. The determined initial phase is determined by the solvent flattening method and histogram matching method using program DM (CCP4 package) or in-phase enhancement program (electron density improvement program). having by matching method The solvent region in the levamylase crystal is 30 to 50%, preferably 35%, and the phase is calculated gradually from low resolution to high resolution to improve the phase with high reliability. 30-60% of the volume is occupied by solvent molecules other than protein (mainly water molecules) In this specification, the volume occupied by solvent molecules in a solution is called a "solvent region." If the crystal has non-crystalline symmetry, it is possible to increase the reliability of the phase by averaging electron density, called non-crystallographic symmetry (NCS) averaging. From the density measurement, we know that two molecules are included in the asymmetric unit The decarbamylase molecule in the crystal is estimated to have an amorphous two-fold axis. Non-crystallographic symmetry including translation and rotation from the electron density map calculated from the phase after applying the solvent smoothing method and the refined heavy atomic coordinates matrix is calculated. At the same time, the area | region in which the protein molecule called a mask exists is identified from the electron density diagram obtained by the solvent smoothing method. The NCS averaging calculation is performed using a non-crystalline symmetric matrix and a mask by program DM or the like to improve the phase with high reliability to obtain an electron density diagram used for constructing a three-dimensional structure model.
[38] The three-dimensional structural model of decarbamylase can be constructed in the following order from the electron density diagram displayed on three-dimensional graphics by program O (A. Jones, Uppsala Universitet, Sweden). First, a plurality of regions (partial sequences including tryptophan residues) having characteristic amino acid sequences are started to be found on the electron density diagram. A partial structure of amino acid residues suitable for electron density is constructed on the three-dimensional graphic using the program O while referring to the amino acid sequence as a starting point of the found region. By repeating this task sequentially, all amino acid residues of decarbamylase are matched to corresponding electron densities to build an initial conformational model of the entire molecule. The three-dimensional coordinates describing the three-dimensional structure are refined according to the refinement protocol of XPLOR (A. T. Brunger, Yale University), which is a structural refinement program, using the constructed three-dimensional structure model as a starting model structure. Also, a decarbamylase mutant having a conformational structure of natural crystals of decarbamylase (for example decarbamylase as shown in SEQ ID NO: 1) and a decarbamylase mutant (for example as shown in SEQ ID NO: 2). The natural crystal conformation of the sieve) is obtained by obtaining the initial phase by molecular substitution using the stereostructure of the obtained EMTS derivative crystal and following the above-described electron density improvement, model construction, and structure refinement. The structure can be determined. This completes the determination of the steric structure of the decarbamylase in the present invention. The enzymes similar in function to decarbamylase are registered in the protein data bank (PDB), a public data bank of the determined decarbamylase and protein conformations. I include it) and a three-dimensional structure. The conformation of N-carbamyl-sarcosine-amidohydrolase, which catalyzes the same decarbamylation reaction as decarbamylase, is known (protein data bank ID). , 1NBA), decarbamylase conformational structure and structural similarity with the conformation of this enzyme is not recognized. Among the proteins whose conformation is registered in the protein data bank, penicillin acylase (1PNK), glucosamine-6-phosphate synthetic enzyme (1GDO), glutamine Phosphoribosylpyrophosphate aminotransferase (1ECF) (glutamine phosphoribosylpyrophosphate amidotransfelase (1ECF)) and proteosomes (1PMA) are included in substructures called domains included in their conformation. And a structure called a four-layer sandwich in which α helix structures are in close contact with both sides of a β sheet laminated in two layers, and have a three-dimensional structure of decarbamylase. The similarity with is recognized. However, in these domain structures and decarbamylase conformations, the geometrical arrangement of α helices and β strands, the so-called topologies of protein conformation (TP Flores et al. (1994), Prot. Eng. 7 , 31-37). In other words, the three-dimensional structure of decarbamylase is characterized by having parallel β-sheets among the β-sheet structures (Fig. 1), and decarbamylase is a new three-dimensional structure even with a protein having a four-layer sandwich structure. As such, "phase" refers to the arrangement or spatial arrangement of secondary structural units of a protein in the present specification.
[39] In the present specification, "α helix" is one of the secondary structures of a protein or polypeptide, and one of the most energy-stable structures having a spiral structure of 5.4 kPa with an amino acid one rotation every 3.6 residues Say. Examples of amino acids that easily form α helices include glutamic acid, lysine, alanine and leucine. On the contrary, as amino acids which are difficult to form α helix, valine, isoleucine, isoproline, proline, glycine and the like can be given. In the present specification, "β sheet" is one of the secondary structures of a protein or a polypeptide, and two or more polypeptide chains having a zigzag-extending structure are arranged in parallel to form an amide group of a peptide. And carbonyl groups, each of which forms a hydrogen bond between the carbonyl group and the amide group of the adjacent peptide chain to form an energy stable sheet. In addition, "parallel β sheet" means that the arrangement direction of the amino acid sequence of adjacent polypeptide chains in (beta) sheet is the same direction, and "antiparallel beta sheet" means the amino acid of the polypeptide chain which adjoins in (beta) sheet | seat. It means that the arrangement direction is reverse. In addition, in this specification, a "(beta) sheet" means the one peptide chain which has a structure extended by the zigzag which forms a (beta) sheet.
[40] About 30 residues of the carboxy-terminal region of decarbamylase (near amino acid position 280 to carboxy-terminal position 303 of SEQ ID NO: 1 or 2) are dimers due to intermolecular interaction. It was found out that the decarbamylase crystals provided in the present study showed that dimers formed tetramers by intermolecular interactions. The fourth four-layer sandwich structure consists of secondary structural units of four α helices and 12 β sheets. Table 1 shows all secondary structural units.
[41] Table 1
[42] Secondary structural units of dicarbamylase
[43] αhelixβ strand (β strand) Arg20-Arg36Glu61-Asp65Arg78-Leu87Glu145-Tyr148Pro177-Leu185Thr211-Asn226Arg279-Arg282Gln3-Gln10Phe41-Val43Gly90-Val199Arg106-Val114Ile120-Arg125Val158-Val161Ala164-Met168Ile190-Tyr195Trp229-Gly234Gly237-Glu239Cys242-Leu244Cys249-Val251Ile257-LeuA260sp274
[44] Hereinafter, the characteristic of the three-dimensional structure of decarbamylase in this invention is put together.
[45] (1) A β sheet composed of six β strands is laminated in two layers, and has a structure called a four-layer sandwich in which two α helices are in close contact with both sides thereof (Fig. 1 and Fig. 1). 2). In Fig. 1, four α-helices (referred to as α1, α3, α5 and α6, respectively) are closely attached to the 1st, 3rd, 5th, and 6th counts from the amino terminus.
[46] (2) The β-sheet is formed of six parallel β-strands and has an orientation not shown in a protein whose conformation is known (Fig. 1).
[47] (3) As amino acid residues catalyzing enzyme reactions, one cystein residue, two glutamic acid residues, one lysine residue, one histidine residue and arginine 2 It forms an active site (two active sites) containing residues (two arginin residues) and having a substrate-binding mode in which the conformation is not known (Fig. 3).
[48] (4) The carboxy-terminal region of about 30 residues forms a dimer structure by intermolecular interaction.
[49] In addition, specific coordinate data of the three-dimensional structure is a compound: DN-carbamoyl-D-amino acid amidohydrolase (PDB, operated by Protein Data Bank, The Research Collaboratory For Structual Bioinformatics (RCSB)) -Carbamyl-D-Amino Acid Amidohydrolase), Exp. Method: It is registered as an X-ray diffraction (registration number: 1ERZ), and this data is used in this specification.
[50] By the completion of the conformational determination of decarbamylase in the present invention, the residues involved in the catalytic activity of the enzyme can be estimated, and not only the stereostructure of the enzyme alone, but also the substrate (for example, DN-carbamoyl) -Three-dimensional structural model of a complex incorporating hydroxyphenylglycine (DN-carbomoyl-hydroxyphenylglycine) molecular modeling method (Swiss-PDBViewer (described above), Autodock (Oxford Molecular), Guex, N.). And Peitsch, MC (1997) SWISS-MODEL and the Swiss-PDBViewer: An environment for comparative protein modeling, Electrophoresis 18, 2714-2723; Morris, G. M et al., J. Computational Chemistry, 19: 1639-1662, 1998; Morris; , GM et al., J. Computaer-Aided Molecular Design, 10, 294-304, 1996; Goodsell, DS et al., J. Mol. Recognition, 9: 1-5, 1996). From this stereostructure and a stereoscopic model, data relating to the structure of the amino acid residues in the catalytic reaction group and the reaction mechanism at the active site can be obtained. The active site of decarbamylase is formed of a cavity containing amino acid residues of Glu46, Lys126, His143, Glu145, Cys171, Arg174 and Arg175 (Fig. 4). Interpretation of Amino Acid Residues Conserved in Glu46, Lys126, Glu145, and Cys171 Found by Comparison of Amino Acid Sequences with Decarbamilases Such as Amidases, Nitrilas, and Decarbamilases It has been suggested from the fact that it is strongly involved in catalysis. In particular, Cys171 is assumed to be an essential catalytic residue for forming an acyl intermediate, an intermediate of the enzymatic reaction (FIG. 3), indicating that decarbamylase is cysteinhydrolase. This fact is consistent with the fact that Cys171 loses catalytic activity in the mutation to Ser171 (R. Grifantini et al. (1996), J. Biol. Chem. 271, 9326-9331). The role of Arg174 and Arg175 contributes to the stabilization of the carboxyl group of the substrate DN-α-carbamyl anino acid by electrostatic interaction. I think.
[51] Based on the facts found in these three-dimensional structures, modifications aimed at improving the stability of decarbamylase or enzymatic activity can be designed. In the present specification, the term "stability" of an enzyme means that the enzyme activity is thermally denatured even after denaturation of the enzyme at a temperature higher than a normal living environment (for example, 70 ° C). At least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90% as compared to before . The improvement of stability can be measured by (DELTA) Tm (difference of denaturation temperature), for example. As used herein, the term "enzyme activity" refers to an activity of converting a D-N-carbamoyl-α-amino acid into a corresponding D-α-amino acid when referring to decarbamylase.
[52] As used herein, the "design method" or "molecular design method" of a mutant molecule refers to an amino acid sequence and a conformational structure of a protein or polypeptide molecule (for example, naturally-occurring molecule) before mutation. By interpreting, it is appropriate to predict what properties each amino acid has (e.g. catalytic activity, interaction with other molecules, etc.) and to modify the desired properties (e.g. improve catalytic activity, improve protein stability, etc.). To calculate amino acid mutations. This design method is preferably done using a computer. Examples of computer programs used in this design method include the following as mentioned herein: DENZO (Mc Science), which is a program for processing X-ray diffraction data, as a program for analyzing a structure; PHASES (Univ. Of Pennsylvania, PA, USA) as a processing program for determining phase; Program DM (CCP4 Package, SERC) as a program for improving the initial phase; Program O (Uppsala Universitet, Uppsala, Sweden) as a program for obtaining three-dimensional graphics; XPLOR (Yale University, CT, USA) as a conformation refinement program; And Swiss-PDBViewer (described above) as a program for modeling mutational influx.
[53] The amino acid mutations used in the design of the mutants herein include, for example, addition, deletion, or modification of amino acids in addition to the substitution of amino acids. have. Substitution of an amino acid means replacing the original peptide with one or more, for example, 1-20, preferably 1-10, more preferably 1-5 amino acids. The addition of amino acids means the addition of one or more, for example 1-20, preferably 1-10, more preferably 1-5 amino acids to the original peptide chain. Deletion of amino acids means the deletion of one or more, for example 1-20, preferably 1-10, more preferably 1-5 amino acids in the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, and amination. Acylation (eg, acetylation) and the like, but is not limited thereto. The amino acid to be substituted or added may be a naturally-occurring amino acid, or may be a non-naturally-occurring amino acid or an amino acid analog. Natural amino acids are preferred.
[54] The term "natural amino acid" means the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine tyrosine, tryptophan, cysteine, proline, histidine, asparatic acid, asparagine, glutamic acid, glutamine, glutamine, γ-carboxyglutamic acid (γ-carboxyglutamic acid), arginine, arginine, ornithine and lysine. Unless otherwise indicated, all amino acids herein are L-isomers.
[55] The term "non-natural amino acid" means an amino acid that is not normally found naturally in protein. Examples of non-natural amino acids include norleucine, paranitrophenylalanine, homophenylalanine, parafluorophenylalanine, 3-amino-2-benzylproprionic acid (3-amino-2 -benzylproprionic acid), homo-arginine D-homoarginine or L-homoarginine (L-homoarginine) and D-phenylalanine (D-phenylalanine).
[56] An "amino acid analog" refers to a molecule which is not an amino acid but is similar in physical properties and / or function of an amino acid. As an amino acid analog, ethionine, canavanine, 2-methylglutamine, etc. are mentioned, for example.
[57] In another embodiment of the invention, the mutants of decarbamylase include ammonium salts (including alkyl or aryl ammonium salts), sulfates, hydrogen sulfates , Phosphate, hydrogen phosphate, dihydrogen 2 phosphate, thiosulfate, carbonate, bicarbonate, benzonate, sulfonate ), Salt forms of peptides such as thiosulfonate, mesylate (methylsulfonate), ethlysulfonate and benzenesulfonate.
[58] Hereinafter, mutation of the protein amino acid for producing a mutant will be described. Methods of performing amino acid substitution and the like include, but are not limited to, changing the codons of DNA sequences encoding amino acids in techniques using chemical synthesis or genetic engineering.
[59] Some amino acids can be substituted with other amino acids in the protein structure such as, for example, cationic regions or binding sites of substrate molecules, without clearly degrading or losing the binding capacity of the interaction. What defines the biological function of a protein is the ability and nature of the protein to interact. Therefore, the substitution of a specific amino acid is made at the amino acid sequence or at the level of the DNA code sequence, and a protein is maintained that retains its original properties even after the substitution. Thus, various modifications can be made in the disclosed peptides or corresponding DNAs encoding the peptides without clearly losing biological utility.
[60] When designing such alterations, the hydrophobicity index of the amino acids (指數 水性 指數) can be considered. The importance of hydrophobic amino acid indexes in assigning interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, R. F. J. Mol. Biol. 157 (1): 105-132, 1982). The hydrophobic nature of the amino acids contributes to the secondary structure of the resulting protein, which in turn is associated with the protein and other molecules (eg enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Define the interaction. Each amino acid is assigned a hydrophobicity index based on the nature of their hydrophobicity and charge. They are: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Asparatic acid (-3.5); Asparagine (-3.5); Lysine (-3.9) and arginine (-4.5).
[61] It is well known in the art that one amino acid can be substituted with another amino acid having the same hydrophobicity index and still generate a protein with the same biological function (e.g., a protein equivalent in enzymatic activity).周知) is true. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5. It is recognized in the art that substitution of such amino acids based on hydrophobicity is efficient. The following hydrophillicity index is assigned to amino acid residues as described in US Pat. No. 4,554,101: arginine (+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5) and tryptophan (-3.4). It is recognized that amino acids can be substituted with other having the same hydrophilicity index and still confer a biological equivalent. In such amino acid substitutions, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.
[62] In the present invention, "conservative substitution" refers to a substitution in which the hydrophilicity index and / or hydrophobicity index between the original amino acid and the amino acid substituted in the amino acid substitution is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitution in each of the following groups: arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and asparagine; Valine, leucine, isoleucine, etc. are mentioned, but it is not limited to these.
[63] In the molecular design aimed at improving the inactivation of enzymes and optimizing the pH of the reaction, information on the catalytic reaction mechanism obtained in the steric structure is very useful. In the present invention, by changing the pKa of the side chain sulfide (SH) group of Cys171 of decarbamylase, amino acid mutations can be designed according to changes in inactivation and optimum pH. Specifically, the effect of amino acid mutations on pKa in SH phase can be determined by electrostatic potential calculation of proteins (Takahashi et al., (1992), Biopolymers 32, 897-909), and the appropriate amino acid mutations can be designed. have. For example, dissociation of the thiol group (SH) of Cys171 by using an electrostatic field near the sulfur atom of Cys171 as a positive electric field. Mutations that promote)) and which do not significantly affect the electrostatic field near the side chain carboxyl groups of Glu46 and / or Glu145.
[64] From the steric structure of decarbamylase in the present invention, five cysteine residues present in decarbamylase are involved in functions and physical properties such as catalytic activity and resistance to air oxidation. It is estimated. Regarding the role of the cysteine residue of decarbamylase, compared with decarbamylase derived from the above strain (Agrobacterium sp. KNK712), it is highly homologous-describe bacterium (配 列 相同) Studies on the decarbamylase derived from another strain (Agrobacterium radiobacter NRRL B11291) with a combination of amino acid mutations, denaturing agents, and Cys modification agents (R. Grifantini et al., (1996), J. Biol. Chem. 271, 9326-9331). In this study, all five residues of cysteine are not involved in the formation of disulfide bonds, and Cys172 of decarbamylase, which is derived from A. radiobacter NRRL B11291, corresponding to Cys171, is substituted with Serine and catalyzed. It is estimated that Cys171 is essential for catalytic activity, although activity is lost, but other cysteine mutations do not affect catalytic activity. This is strongly suggested by the fact that the corresponding cysteine residues are also conserved in other similar enzymes. As used herein, the term "corresponding amino acid" refers to an amino acid that has or is predicted to have the same action as a predetermined amino acid in a protein or polypeptide, which protein or polypeptide is a comparison standard in which protein molecule or polypeptide molecule. In particular, in the enzyme molecule, it refers to an amino acid present at the same position in the active site and making the same contribution to the catalytic activity.
[65] In decarbamylase having the amino acid sequences of SEQ ID NOs: 1 and 2, Cys192 and Cys249 are assumed to be buried in the molecule because they are difficult to undergo chemical modifications, and Cys242 and Cys278 are easily subjected to chemical modifications. Therefore, it is assumed that it is located in the loop structure portion of the molecular surface. A substitution of alanine for cysteine of two residues of A. radiobacter NRRL B11291 (Cys243 and Cys279, respectively) corresponding to Cys242 and Cys278 of SEQ ID NO: 1 yields a mutant having improved stability (Japanese Patent Laid-Open No. 8-84584). As described above, regarding Cys242 and Cys278, it is confirmed from the conformation that they exist on the molecular surface, and it is suggested that these Cys residues are involved in the resistance to deoxidation of decarbamylase. Regarding Cys192 and Cys249 embedded in the molecule, it is believed that it does not contribute significantly to the improvement of resistance to air oxidation, but a larger volume of hydrophobic amino acid residues by complementing the cavity in the side chain volume or molecule ( By creating a mutant that is substituted with hydrophobic amino acid), decarbamylase can be produced that improves stability against heat or organic solvents.
[66] In addition to the cysteine residues, the involvement of methionine residues can be considered in lowering enzyme activity and conservation of stability due to air oxidation of decarbamylase. From the three-dimensional structure of the decarbamylase in the present invention, five residues of the methionine residues in the decarbamylase were completely buried inside the molecule, while two residues completely exposed to the outside of the molecule (Met238 and Met243) is present in the turn structure portion, and these two residues are believed to be deeply related to the resistance of decarbamylase to air oxidation. As used herein, the term "turn structure" or "β turn structure" refers to a local structure consisting of three or more amino acid residues that greatly change the direction of propagation of a peptide chain between secondary structures in a three-dimensional structure of a protein. (local structure: 局 所 構造). In addition, since Met4 and Met72 are located near the surface of the molecule and are usually buried inside the molecule but easily accessible to solvent molecules and the like, they may be involved in decarbamylase resistance to air oxidation. have. Based on the results of these structures, it is possible to design a separate amino acid substitution that is expected to improve decarbamylase resistance to air oxidation. In other words, improved resistance can be achieved by making a mutant that replaces Met4 and Met72 with a side chain volume or a cavity in the molecule and also replaces hydrophobic amino acid residues which are not subjected to air oxidation. Since Met238 and Met243 are completely exposed to the outside of the molecule, it is preferable to improve the resistance by replacing these residues with neutral or hydrophilic amino acids which are not subjected to air oxidation. In addition, since the turn structure part composed of the regions of the amino acids Met238 to Met243 of decarbamylase also contains Cys242, which is weak in air oxidation, a triple mutant is substituted to replace these three residues with an appropriate amino acid. Or resistance to air oxidation can be improved by making a mutant that deletes a turn structure containing three residues. In addition, resistance to air oxidation can be improved by replacing the turn structure containing these residues with the β turn structure of other proteins. In mutant design of cysteine and methionine, optimal amino acids can be selected by mutating these residues on computers with other amino acids and interpreting the structural energy with respect to the stability of the mutant.
[67] In addition to cysteine residues and methionine residues, amino acid mutations that complement the cavities in the protein molecule (e.g., the cavity formed near amino acid Asn92 of decarbamylase), amino acids with energetic disadvantageous structures (e.g. decarbamylase) Designs focusing on stabilizing factors of commonly known proteins, such as mutations in amino acids Pro203 and Val236) and stabilization of the α helix structure, can also be designed.
[68] As a design strategy that is different from the alteration design by the amino acid mutation, stability is improved by modifying decarbamylase to a lower molecular weight enzyme by deleting a region that is not considered to be involved in the enzymatic activity of decarbamylase. Can be improved. For example, it can be modified to a denser decarbamylase by deleting part or all of the loop-shaped structural region at a position away from the active site. It was also estimated by the present inventors that the area of about 30 residues at the carboxy-terminus of decarbamylase is involved in the dimer formation. Decarbamylase is assumed to exist as a dimer or tetramer when exhibiting catalytic properties in a general buffer. On the other hand, it is assumed that the monomer protein is advantageous in the process of recovering from the denatured state of the protein. Deleting some or all of the carboxy-terminal about 30 residue region can prevent dimer formation, thereby preparing a stable monomer. Industrially, decarbamylase is used as a so-called immobilized enzyme immobilized on a carrier, but even in this case, the monomer can achieve more efficient immobilization.
[69] The decarbamylase mutant in the present invention can be designed or prepared by various methods in addition to the above-described methods. For example, an array of enzymes having decarbamylase activity may be used for oligonucleotide-specific mutagenesis or other conventional techniques at sites that are preferably identified for mutations using the present invention. It may be mutated by technical (eg deletion) means. Alternatively, mutants of decarbamylase can be made by site-specific substitution of non-natural amino acids of specific amino acids. For example, decarbamylase mutants can be made by substitution of certain cysteine or methionine residues with selenocysteine or selenomethionine. It depletes either (or both) of natural cysteine or methionine, and is a wild-type polypeptide on a growth medium richly supplied with selenocysteine or selenomethionine (or both) or It can be achieved by propagating a host organism capable of expressing any of the mutant polypeptides. When using the homologous recombinant method, mutations may be introduced into a DNA sequence encoding decarbamylase using a synthetic oligonucleotide. These oligonucleotides comprise a nucleotide sequence adjacent to a desired mutation site. Mutations can be made in the full-length DNA sequence of decarbamylase or any sequence encoding its fragment polypeptide.
[70] Mutant decarbamylase DNA sequences produced by this method or by alternative methods known in the art can be expressed using expression vectors. As is well known in the art, expression vectors are typically elements that enable self replication in a host cell that is independent of the host genome. and one or more phenotype markers for selection purposes. Expression vectors prior to or after insertion of the DNA sequence surrounding the coding sequence of the desired decarbamylase mutant may be selected from promoters, operators, ribosomal binding sites, translation initial signals, and needs. And a control sequence encoding a repressor gene or various activator genes and stop signals. In some embodiments, nucleotides encoding a "signal sequence" can be inserted before the decarbamylase mutant code sequence if desired secretion of the mutants produced. To express in a controlled state of the regulatory sequence, the desired DNA sequence must be operably linked to the control sequence. That is, the sequence encoding the decarbamylase mutant in the controlled state of the control sequence must have a start signal (ie ATG) to maintain an appropriate reading frame in order to be able to produce the expression product of that sequence. .
[71] All well-known and available expression vectors are useful for expressing the mutated decarbamylase code sequence in the present invention. These include yeast plasmids such as known bacterial plasmids (eg pBR322), plasmids of wider host regions (eg RP4, phage DNA), 2μ plasmids or derivatives thereof, and Chromosomal DNA sequences such as vectors capable of combining plasmid and phage DNA, non-chromosomal DNA sequences and fragments of synthetic DNA sequences.
[72] In addition, any of a wide range of expression control sequences that control expression when operably linked to a DNA sequence are used among these vectors to express the mutated DNA sequence according to the present invention. Such useful expression control sequences include, for example, other arrangements and combinations thereof that are known to control viral gene expression.
[73] Hosts of a wide variety of species are useful for the production of decarbamylase mutants according to the invention. As these hosts, for example, bacteria such as E. coli, Bacillus and Streptomyces, fungi such as yeast, animal cells such as CHO cells, plant cells And transgenic host cells.
[74] Expression vectors and expression systems express mutant DNA sequences in the present invention and produce decarbamylase mutants do not necessarily function in the same manner. Not all hosts use the same expression system to function well. However, one of ordinary skill in the art can select appropriate vectors, expression regulatory sequences and hosts without departing from the scope of the present invention without much experimentation.
[75] For example, when making a selection, the replication ability of the vector should be considered. Various factors should be considered when selecting an expression control arrangement. These include, for example, the relative strength of the system, its controllability, its compatibility with the DNA sequence encoding the decarbamylase mutants in the present invention, and in particular with regard to potential secondary structures. It should also be considered. The host is compatible with the selected vector, the toxicity of the decarbamylase mutant to the host, the ability to secrete the mature product, the ability to fold the protein properly, and the appropriate higher order structure. It should be selected by consideration of its ability to form high fermentation requirements, fermentation requirements, ease of purification of decarbamylase mutants in the host, and safety. Among these parameters, one of skill in the art can select various vector / expression control system / host combinations that can produce useful amounts of mutant decarbamylase.
[76] Decarbamylase mutants produced in these or other systems can be purified by a variety of conventional processes, including those used to purify natural enzymes having decarbamylase activity.
[77] Once the mutation to decarbamylase occurs at the desired location (eg, active site, stability site or binding site, etc.), the resulting mutant can be tested for any of several properties of interest.
[78] For example, mutants can be screened for charged changes in physiological pH. This is determined by measuring the isoelectric point of the decarbamylase mutant compared to the isoelectric point (pI) of the decarbamylase before mutation. The isoelectric point is Wellner, D., Analyt. Chem., 43, p. It is measured by gel electrophoresis by the method of 597 (1971). The mutant whose surface charge has changed is a decarbamylase protein having a pI changed by a substituted amino acid located on the surface of the enzyme as provided by the structural information in the present invention.
[79] Mutants can also be screened for high inactivity compared to decarbamylase prior to mutation. The activity of the mutant can be determined by, for example, DN-carbamoyl-α-amino acid using D-α-amino acid using the assay table described herein (see Example 7 below). This is determined by measuring the ability to convert to a stream.
[80] The D-α-amino acids produced using the decarbamylase mutants in the present invention are pharmaceutical intermediates (e.g., D-phenylglycine and D-para-hydroxyphenylglycine ( D-para-hydroxyphenlyglycine), which can be used for the manufacture of medicines (eg, synthetic penicillin and synthetic cephalosporin). Pharmaceutical compositions containing a medicament thus prepared may also be provided by the present invention. The pharmaceutical composition may contain any pharmaceutically acceptable auxiliary ingredient, including excipients, stabilizers, carriers and the like.
[81] D-α-amino acids produced using the decarbamylase mutant of the present invention are used as pesticide intermediates (e.g., D-valine) and pesticides (e.g. fluvalinate). It can be used for the production of)). A pesticide composition containing the pesticide thus prepared may also be provided by the present invention. The agricultural composition herein may contain any agriculturally acceptable auxiliary ingredient such as excipients, stabilizers, carriers and the like.
[82] D-α-amino acids produced using the decarbamylase mutants of the present invention are intermediates of food additives (for example, D-alanine and D-asparagine acid). As an additive, it can be used for the production of food additives (e.g., alitame).
[83] In another embodiment of the present invention, inhibitors of decarbamylase mutants or similar enzymes can be designed and prepared. Reaction inhibitors can be designed using structural information of decarbamylase. Those skilled in the art will appreciate, for example, that the inhibitor is competitive by computer compatible enzyme reaction kinetic data using standard formulas by Segel, IH, Enzyme Kinetics, J. Wiley & Sons, (1975). It can be identified as being competitive, uncompetitive, or noncompetitive. It is also possible to design reaction inhibitors using information of reaction intermediates of decarbamylase or its analogues (eg complex conformation with substrates or reaction products). This information is useful for the design of improved analogs of known compounds and for the design of new classes of reaction inhibitors as known decarbamylase or analogue enzyme inhibitors thereof.
[84] In another separate embodiment of the present invention, a polypeptide enzyme or protein enzyme similar to the amino acid sequence of decarbamylase (e.g., amidase or nitrile) using the steric structure of decarbamylase in the present invention. Aze) can be modified. Modifications can be made in the same way as the design of the mutants described above. Here, the "similar polypeptide enzyme or protein enzyme" is preferably at least 30%, preferably with respect to the amino acid sequence of the decarbamylase and its full length amino acid sequence whose amino acid sequence is shown in SEQ ID NO: 1 or 2. Preferably at least 50%, more preferably equal to at least 80%, in particular the region defining the active site of decarbamylase (amino acid number 38 comprising amino acids Glu46, Lys126, Glu145 and Cys171 of SEQ ID NO: 1 or 2). Typically at least 60%, preferably at least 80%, more preferably 90%, even more preferably 95% with respect to amino acids in the range of -49, 108-127, 143-148 and 164-177). same.
[85] Examples of transforming properties of an enzyme using similar enzyme conformations include the conformation of isopenicillin synthetic enzymes with a substrate specific transformation and analogous structure of an enzyme called expandase. There is research using. See pamphlet of WO97 / 02005. In this document, it is suggested that the conformation is similar to the isopenicillin synthase (Roach (1995), Nature, 375, pages 700 to 704) and the primary structure (amino acid sequence), and the conformation is similar. Mutant enzymes that act on penicillin G by identifying amino acid residues that are involved in substrate recognition of an expandase are identified by sequence comparison of both enzymes, and by transforming the substrate specificity by mutation of their amino acid residues. Succeeded in making.
[86] The identity comparison of amino acid sequences can be calculated using, for example, the following sequence analysis tool: FASTA (W. R. Pearson and D. J. Lipman, PNAS85, 2444-2448 (1988)); BLAST (S. F. Altschul, T. L. Madden, A. A. Schaffer, J, -H., Zhang, Z. Zhang, W. Miller and D. J. Lipman (1997) Nucl. Acids. Res. 25: 3389-3402 (1997)); Unix-base GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Madison, Wisconsin, USA53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB101RQ, England) and the Ex PASy World Wide Web molecular biology server (Geneva University Hospital and University of Geneva, Geneva, Switzerland) and MacVector 6.0 (Teijin system technology).
[87] According to the present invention, a mutant of a polypeptide enzyme or protein enzyme similar to the amino acid sequence of decarbamylase obtained using the above method can be provided. Mutants of these enzymes may be used instead of the wild type enzyme, for example as a bioreactor or the like.
[88] In another aspect, the present invention provides a system for designing decarbamylase mutants using a computer. This system designs a decarbamylase mutant that improves physical properties and / or functions based on the determined conformation and means for determining the conformation of enzyme crystals having decarbamylase activity by X-ray crystal structure analysis. Means for doing so. The computer system according to the present invention can be constructed using a computer system known in the art, for example, a system known in the art as described herein.
[89] According to the present invention, a three-dimensional coordinate data of the molecule such as decarbamylase and / or a program of a molecular design method and / or a modification method can be recorded and a computer-readable recording medium can be provided. have. As a computer-readable recording medium, for example, a magnetic tape (floppy disk, etc.), a magneto-optical disk (MO, etc.), an optical disk medium (CD-ROM, CD-R, CD-RW, DVD-ROM, etc.) Can be mentioned. In one embodiment, such computer readable recording medium is a computer readable recording medium having recorded thereon a program for carrying out the design process of the decarbamylase mutant. Here, this design treatment is a decarba which improves physical properties and / or functions based on the process of inputting the data of the crystal determined by the X-ray crystal structure analysis of the enzyme crystal having decarbamylase activity and the determined conformation. It may include a process for designing a milase mutant. In another embodiment, the present invention provides a recording medium for recording data describing the conformation of the decarbamylase mutant. Here, the conformation of this mutant is a process of inputting the data of the crystal determined by X-ray crystal structure analysis of an enzyme crystal having decarbamylase activity and the determined conformation It can be obtained by a treatment including a step of designing a decarbamylase mutant with improved physical properties and / or function.
[90] In the following Examples, the present invention will be described in more detail, but these are provided for the purpose of illustrating the present invention and do not limit the present invention.
[91] (Example)
[92] Reagents used in the following examples are obtained from Nacalai Tesque, Wako Pure Chemical Industries, Ltd. except as noted. Or SIGMA (St Louis, MO, USA).
[93] Example 1
[94] Preparation of natural crystals of decarbamylase
[95] Decarbamylase was prepared by a known method (see H. Nanba et al., Biosci. Biotechno. Biochem. 62, 875-881 (1998)), at a concentration of 10-20 mg / ml. 0.1 M HEPES buffer (pH 7.5) containing 10 μl of decarbamylase solution (0.001 M HEPES buffer, pH 7.5) and 15-20 wt% polyethylene glycol 6000 (manufactured by Nacalai Tesque) and 0.2 M lithium chloride as precipitant. 10 μl of SIGMA Co., Ltd.) was mixed on a droplet stage, and 300 μl of the solution having the precipitant composition was sealed as a solution bath solution, followed by vapor diffusion method at 20 to 25 ° C. Crystallize with. After initiation of crystallization, crystals grew from about 2 days to 2 weeks in dimensions ranging from 0.3 × 0.3 × 0.1 mm to 0.6 × 0.6 × 0.3 mm.
[96] Example 2
[97] Preparation of Crystals of Heavy Atom Derivatives of Decarbamylase
[98] 20 weights of the natural crystals obtained in Example 1 were removed from the droplets on the droplet stage using a microscope to prepare a concentration of 0.5 to 1.0 mM of EMTS (sodium ethylmercurithiosalicylate), one of the mercury compounds. A heavy atom derivative is prepared by immersing in 0.1 M HEPES buffer (pH 7.5) containing% polyethylene glycol 6000 (made by Nacalai Tesque) and 0.2 M lithium chloride overnight.
[99] Example 3
[100] Freezing Decarbamylase Crystals
[101] Natural crystals and heavy atom derivative crystals of decarbamylase prepared in Examples 1 and 2 were removed from crystallized droplets without adding a freeze stabilizer such as glycerol and directly immersed in liquid nitrogen to freeze them instantly. .
[102] Example 4
[103] Collection of X-ray Diffraction Data of Decarbamylase Crystals
[104] Diffraction data were measured with the SPring-8 facility for radiation analysis by the multi-wavelength anomalous dispersion method (multi-wavelength anomalous dispersion method). The measurement was carried out using crystals of EMTS derivatives frozen in liquid nitrogen. The measurement was carried out at three wavelengths (0.98, 1.00, 1.27 Hz) in consideration of the ideal dispersion of mercury. All three wavelengths of diffraction data could be collected from one crystal. Table 2 shows the results of processing the 53 diffraction image data measured by the data processing program DENZO (Mc Science). Regarding the natural crystals of decarbamylase, diffraction data were measured in a frozen state using a wavelength of 1.00 Hz, and the results of the treatment are shown in Table 3. In addition, the terms of independent reflection, unit lattice constant, and lattice constant are referred to the introductory X-ray analysis (Tokyo Chemical Co., Ltd., such as Masato Kakuto).
[105] Table 2
[106] Diffraction Data Processing Results of EMTS-Induced Crystals of Dicarbamylase
[107] WavelengthResolutionR-merge (%)yield(%)Independent reflectionUnit lattice constant 0.981.85.198.556679a = 66.92b = 135.50c = 67.30 1.001.84.798.557493a = 67.23b = 136.13c = 67.65 1.272.05.886.536870a = 67.13b = 136.00c = 67.54
[108] R-merge represents the error between each frame of multiple measurements.
[109] Table 3
[110] Diffraction Data Processing Results of Natural Crystals of Dicarbamylase
[111] WavelengthDivisionR-merge (%)yield(%)Independent reflectionLattice constant 0.931.76.496.168596a = 67.84b = 137.83c = 68.39
[112] R-merge represents the error between each frame of multiple measurements.
[113] Example 5
[114] Determination of heavy atomic coordinates and phase improvement
[115] Of the three-wavelength diffraction data measured for the EMTS derivative crystal, 1.00 Å data is assumed to be natural crystal data, and 0.98 Å is assumed to be anomalous data and 1.27 Å as isomorphous data. Compute the difference patterson function and the anamorphic difference patterson function and call it the Hacker plane (Fig. 5 to Fig. 7) by drawing the peak position on the Hacker plane with strong intensity and its Identification of the heavy atom positions was attempted from the prominent peak positions corresponding to the cross vectors. The two patterned mercury atoms (mercury 1 and mercury 2) were picked from two difference Patterson plots (between 1.00-0.98 kV and between 1.00-1.27 kV) between the strong Patterson peaks in each hacker plane. Was identified and its coordinates were obtained. Confirmation is to first calculate the phase using only mercury 1 coordinates, and then calculate the mercury 2 coordinates from the difference Fourier calculation using the phases, so that the mercury 2 self peak and mercury 1-are calculated. This is done by determining whether a cross peak of mercury 2 is present in the difference Patterson plot. In order to find the positions of the combined mercury atoms, the coordinates of mercury 1 and 2 were used to refine and phase-enumerate the position parameters of the heavy atoms, and their positions were obtained by differential Fourier calculation to identify mercury 2. As in the case, magnetic peaks and cross peaks were confirmed. As a result, magnetic peaks and cross peaks corresponding to four mercury atoms can be identified, and coordinates of mercury 3, mercury 4, mercury 5, and mercury 6 were determined.
[116] Six mercury atom coordinates (heavy-atomic parameters), which were obtained using 1.8 Å resolution of EMTS derivative crystals measured at a wavelength of 1.00 Å, were refined using the program MLPHARE (CCP4 package) (SERC) to obtain the initial phase. Decided. The mean figure of merit after refinement was 0.50. Thereafter, the solvent flattening method and histogram matching method using the program DM (CCP4 package, SERC) were used as the solvent region in the decarbamylase crystals at 35%. The initial phase was improved by gradually expanding the phase from (低 分解 能) to high resolution. In addition, the crystals of decarbamylase were found to contain two molecules in the asymmetric unit from the density measurement results of the crystal, and the phase improvement was performed by averaging electron densities called NCS averaging using non-crystalline symmetry. First, an amorphous 2fold axis in the decarbamylase crystals was determined to apply this method. Calculate the electron density diagram using the phase after applying the solvent smoothing method, and display the heavy atom position and the electron density diagram on the three-dimensional graphic, and connect the two points between the coordinates of the mercury atom coordinates. We know that we are orthogonal on this Z axis (one of the crystallographic double axes). Thus, decarbamylase molecules are assumed to have three pieces of symmetry, two of which are non-crystalline double axes, and one of which is crystallographic double axes 222. Each section of the electron density diagram was observed in detail to determine the central coordinates of the two molecules, respectively, to produce a non-crystalline symmetric matrix including translation and rotation. At the same time, the area | region in which the protein molecule called a mask exists was identified from the electron density diagram obtained by the solvent smoothing method. Using the obtained non-crystalline symmetry matrix and mask, NCS averaging calculations were performed using the program DM. As a result, the correlation coefficient between the two molecules was 0.92, indicating a high correlation, and the free R-factor was 24.5%. The improved electron density diagram is very clear (see, for example, Fig. 8. In Fig. 8, the relationship of the peptide chain is observed as a continuous electron density, and in the center of the figure, Glu at position 46, position 171 Electron densities corresponding to the amino acid side chains identified by Cys and Glu at position 145 are observed) so that secondary structures such as α helices or β sheets can clearly track the flow of the amino acid main chain In addition, electron densities such as aromatic amino acid side chains were also clear.
[117] Example 6
[118] Construction and refinement of three-dimensional model of decarbamilase
[119] From the electron density diagram obtained in Example 5, a three-dimensional model of decarbamylase was assembled on three-dimensional graphics using the program O (Uppsala Universitet). In order to correspond the electron density to the amino acid sequence, first, find the electron density corresponding to the partial amino acid sequence having characteristic electron density such as tryptophan residue on the electron density diagram and refer to the amino acid sequence to find the partial structure of the amino acid residue suitable for the electron density. The program O was used to build and repeat this work sequentially to match all amino acid residues (303 residues) of decarbamylase to the corresponding electron densities to build up the initial conformational model of the entire molecule. Using the obtained three-dimensional structure model as a starting model structure, the three-dimensional structure was refined according to XPLOR's protocol (XPLOR manual, Yale University). The precision is determined by modifying the local structure, which is largely out of the electron density diagram, and by identifying the electron density corresponding to the water molecule, and repeating the operation including the water molecule in the precision calculation. The free R-factor was refined to an index. The R value of the final model structure in the natural crystals added with water molecules was 19.6% with respect to the reflection data with a resolution of 500-1.7 kHz.
[120] Example 7
[121] Stabilized design of decarbamylase
[122] As an example of stabilization design of decarbamylase, the stability is improved by mutating amino acid residues which are disadvantageous in terms of energy. Proline backbone (dihedral angle: φ-60 °, ψ-44 °) of position 203 and Pro203 calculated from the solid structure in the present invention are characterized by the backside angle characteristic of α-helical structure. Equipped. In general, proline residues are known to act as residues that destabilize or destroy the helical structure. The presence of proline at position 203 of decarbamylase is thought to cause distortion in the main chain structure and thus destabilize the structure. Since this site has a backside angle characteristic of the helical structure, the structural energy in the natural state can be stabilized by substituting the amino acid residue suitable for the helical structure. For example, as an amino acid residue which tends to have an alpha helical structure, it can be substituted with alanine (Ala), glutamic acid (Glu), leucine (Leu), serine (Ser) and the like. The amino acid at position 203 was mutated to 20 types of natural amino acids, and for each structure, the potential parameters of AMBER (Weiner, PA et al., J. Comp. Chemistry 7 (2): 230-252, 1986) ), The structural energy was calculated, and the optimal amino acid mutation at this site was determined as an amino acid mutation having a low structural energy value is a more preferable mutation. The result was asparagine> threonine> serine> valine> glutamic acid> isoleucine> alanine> phenylalanine> tyrosine> leucine> histidine> asparagine> glutamine> cysteine> proline. The denaturation temperature of the amino acid mutant prepared in Example 1 is shown in Table 4 (International Publication No. WO94 / 03613). The improvement of stability (ΔTm = 3.4-8.2 ° C.) was recognized in all the following mutations. It is reasonable to think that these have eliminated distortion of the main chain structure by mutating to amino acids other than proline. In addition, mutants with glutamic acid (ΔTm = 8.2 ° C.) showed the highest stability, but this resulted in the guanidino group and ions of the arginine side chain at position 139 where the glutamic acid side chain carboxyl group was near by the glutamic acid mutation. It is believed that they can form bonds or hydrogen bonds and thus exhibit high stability compared to other mutants due to the contribution of newly increased interactions. The increase in stability of the His mutant is small compared to other mutants with ΔTm = 3.4 ° C., indicating that electrostatic repulsion with arginine at position 139 when histidine has a static charge near pH 7.0. It is considered that the contribution to stabilization is reduced by eliminating the distortion of the main chain.
[123] The stability ranking of the optimal amino acid mutation at position 203, calculated by calculation, is not completely consistent with the magnitude of the increase in denaturation temperature obtained by experiments, but all have lower structural energy (stable than proline residues of native decarbamylase (stable). The upper part was counted from the) side and was in good agreement with the experimental results.
[124] Table 4
[125] Changes in thermostability with amino acid mutations at position 203
[126] MutantMutation positionAmino acid mutationsDenaturation temperature (℃)ΔTm 404Pro203Leu68.06.2 406Pro203Ser66.54.7 429Pro203Glu70.08.2 445Pro203Thr67.55.7 468Pro203Ala67.75.9 469Pro203Ile67.25.4 470Pro203His65.23.4
[127] The denaturation temperature is defined as the temperature at which 50% of the remaining inactive activity is defined by measuring the activity after removing the insoluble material due to heat treatment and thermal denaturation for 10 minutes at the prepared (raw) enzyme solution at each temperature. do. The measurement of remaining inactivity was carried out as follows. 0.1 ml of enzyme solution was added to 1 ml of substrate solution in which DN-carbamoyl-parahydroxyphenylglycine was dissolved in 0.1 M phosphate buffer pH 7.0 so as to be 1.0% by weight. , 0.25 ml of 20% trichloroacetic acid was added to stop the enzymatic reaction, and then D-phenylalanine was used as the internal standard using DN-carbamoyl-parahydroxyphenylglycine, which converted the activity. This was calculated by quantitative determination by high performance liquid chromatography. The mutant strain shows the identification number of the mutant strain obtained by screening. ΔTm represents the difference from the denaturation temperature of decarbamylase before mutation.
[128] According to the present invention, there are provided conformations of decarbamylase and decarbamylase mutants and conformational models of mutants. The conformational structure of this enzyme is useful for rational molecular design of amino acid mutations related to stability of heat resistance, resistance to organic solvents, resistance to air oxidation, changes in pH and optimum activity of the enzyme. As a result, it is possible to obtain a modified enzyme which is advantageous for industrial use quickly and efficiently. In addition, the three-dimensional structure of the enzyme is uniquely determined among a group of enzymes such as amidase or nitrilaase having similar relations, and the three-dimensional structure of these enzymes can be meaningfully applied in industrial applications. Obvious.
[129] (SEQUENCE LISTING)
[130] <110> Kaneka Corporation
[131] <120> Three-dimensional structure of decarbamilase and the use
[132] <130> F5-00PCT217 / KA022PCT
[133] <140> ND
[134] <141> 2000-8-28
[135] <150> JP11-246797
[136] <151> 1999-08-31
[137] <160> 2
[138] <170> Patent In Ver. 2.1
[139] <210> 1
[140] <211> 303
[141] <212> PRT
[142] <213> Agrobacterium sp.
[143] <400> 1
[144] Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala Arg
[145] 1 5 10 15
[146] Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Asp Met Leu Thr
[147] 20 25 30
[148] Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu Ala
[149] 35 40 45
[150] Leu Thr Thr Phe Phe Pro Arg Trp His Phe Thr Asp Glu Ala Glu Leu
[151] 50 55 60
[152] Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro Leu
[153] 65 70 75 80
[154] Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr Ala
[155] 85 90 95
[156] Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser Ile
[157] 100 105 110
[158] Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile His
[159] 115 120 125
[160] Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His Leu
[161] 130 135 140
[162] Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr Asp
[163] 145 150 155 160
[164] Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg Trp
[165] 165 170 175
[166] Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile Cys
[167] 180 185 190
[168] Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His Asp
[169] 195 200 205
[170] His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser Tyr
[171] 210 215 220
[172] Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Val Gly Met Glu Glu
[173] 225 230 235 240
[174] Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly Glu
[175] 245 250 255
[176] Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala Ala
[177] 260 265 270
[178] Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn Phe
[179] 275 280 285
[180] Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu
[181] 290 295 300
[182] <210> 2
[183] <211> 303
[184] <212> PRT
[185] <213> E. coli
[186] <400> 2
[187] Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala Arg
[188] 1 5 10 15
[189] Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Asp Met Leu Thr
[190] 20 25 30
[191] Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu Ala
[192] 35 40 45
[193] Leu Thr Thr Phe Phe Pro Arg Trp Tyr Phe Thr Asp Glu Ala Glu Leu
[194] 50 55 60
[195] Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro Leu
[196] 65 70 75 80
[197] Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr Ala
[198] 85 90 95
[199] Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser Ile
[200] 100 105 110
[201] Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile His
[202] 115 120 125
[203] Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His Leu
[204] 130 135 140
[205] Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr Asp
[206] 145 150 155 160
[207] Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg Trp
[208] 165 170 175
[209] Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile Cys
[210] 180 185 190
[211] Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Glu Val Pro Gln His Asp
[212] 195 200 205
[213] His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser Tyr
[214] 210 215 220
[215] Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Ala Gly Met Glu Glu
[216] 225 230 235 240
[217] Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly Glu
[218] 245 250 255
[219] Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala Ala
[220] 260 265 270
[221] Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn Phe
[222] 275 280 285
[223] Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu
[224] 290 295 300

权利要求:
Claims (35)
[1" claim-type="Currently amended] Amino acid sequence or tetragonal space represented by the space group P2 ₁ 2 ₁ 2 and ordinal number (SEQ ID NO.) 1 of the orthorhombic system. A decarbamylase crystal comprising the amino acid sequence represented by group P2 ₁ 2 ₁ 2 ₁ and SEQ ID NO: 2.
[2" claim-type="Currently amended] The method of claim 1,
The crystal has a unit cell having a rectangular parallelepiped shape, and has a lattice constant: a = 66.5 to 68.5 kPa, b = 135.5 to 138.0 kPa, c = 66.5 to 68.5 kPa, and said amino acid sequence is SEQ ID NO: 1, The decarbamylase crystal | crystallization characterized by the above-mentioned.
[3" claim-type="Currently amended] The method of claim 1,
The crystal has a rectangular lattice shape, has a unit lattice constant: a = 68.5-70.5 ms, b = 138.0-140.5 ms, c = 68.5-73.0 ms, and the amino acid sequence is SEQ ID NO: 1 Decarbamylase crystals.
[4" claim-type="Currently amended] The method of claim 1,
Wherein the crystal has a rectangular lattice shape, has a unit lattice constant: a = 81.5 to 82.5 ms, b = 133.0 to 135.0 ms, c = 119.5 to 121.5 ms, and the amino acid sequence is SEQ ID NO: 2 Decarbamylase crystals.
[5" claim-type="Currently amended] The method according to any one of claims 1 to 4,
Wherein said crystal comprises at least one heavy metal atom per molecule of decarbamylase.
[6" claim-type="Currently amended] The method of claim 5,
Wherein the heavy metal atom is one of mercury, gold, platinum, lead, iridium, osmium, and uranium.
[7" claim-type="Currently amended] A frozen crystal, wherein the decarbamylase crystal according to any one of claims 1 to 6 is prepared by freezing in a liquid nitrogen state.
[8" claim-type="Currently amended] Supplying decarbamylase solution at a concentration of 1 to 50 mg / ml, containing polyethylene glycol (PEG) or methoxy polyethylene glycol (PEGMME) at a concentration of 5 to 30% by weight and also having a pH A step of supplying a precipitant solution containing a buffer having a concentration of 6.0 to 9.0, a step of mixing the decarbamylase solution with the precipitant solution, and a resultant mixed solution of the decarbamylase in the solution. A process for producing decarbamylase crystals, comprising the step of standing still for a predetermined period until the crystals grow to a predetermined size or more.
[9" claim-type="Currently amended] The method of claim 8,
The mixing step includes mixing droplets of the decarbamylase solution with droplets of the precipitant solution, wherein the standing step is to mix the precipitant solution in a closed container with the mixed droplets obtained in the mixing step. Suspending on a receiving solution reservoir, wherein the vapor pressure of said precipitant solution in said solution tank is lower than the vapor pressure of said mixed droplets.
[10" claim-type="Currently amended] The method of claim 8,
The mixing step includes mixing the droplets of the decarbamylase solution with the droplets of the precipitant solution, and wherein the settling step includes the mixed droplets obtained in the mixing step in a closed vessel containing the precipitant solution in a sealed container. suspending in a droplet stage, wherein the vapor pressure of said precipitant solution in a solution bath is lower than the vapor pressure of said mixed droplets.
[11" claim-type="Currently amended] The method of claim 8,
The period of time for allowing the mixed solution to stand is 1 day to 3 weeks.
[12" claim-type="Currently amended] The method of claim 8,
And disposing the decarbamylase solution in a size exclusion semi-permeable membrane after the step of supplying the solution of decarbamylase, wherein the mixing step comprises depositing a precipitant solution into the semipermeable membrane. Diffusing into the decarbamylase solution by passing through the same.
[13" claim-type="Currently amended] The method of claim 8,
And said step of mixing comprises slowly adding the precipitant solution to the decarbamylase solution, and wherein the step of standing still includes placing the obtained mixed solution in a sealed container.
[14" claim-type="Currently amended] Decarbamylase or an active fragment thereof characterized by a conformation having a conformational topology of the protein shown in the figures below:

[15" claim-type="Currently amended] A decarbamylase or active fragment thereof, comprising a four-layer sandwich structure comprising a secondary structure comprising four α helixes and 12 β sheets.
[16" claim-type="Currently amended] The amino acid residues involved in the enzymatic reaction are one cystein residue, two glutamic acid residues and one lysine residue. DN-carbamoyl-α-amino acid, decarbamylase or decarbamila, characterized by the steric structure of the active site with the substrate binding mode shown in the following figure. Mutants or active fragments thereof:

(Herein, substituent R is a side chain of D-N-carbamoyl-α-amino acid.)
[17" claim-type="Currently amended] An enzyme molecule having decarbamylase activity based on DN-carbamoyl-α-amino acid, wherein the amino acid at least in SEQ ID NO: 1 or 2 is: Glu at position 46, Lys at position 126, position 145 An enzyme molecule or an active fragment thereof having an active site cavity formed from an amino acid corresponding to Glu and Cys in position 171.
[18" claim-type="Currently amended] The method of claim 17,
In the active site cavity, when the DN-carbamoyl-α-amino acid reacts, Lys at position 126, His at position 143, Glu at position 145, Arg at position 174, position at position 1 or 2 An enzyme molecule or an active fragment thereof, capable of interacting with an amino acid corresponding to Arg at 175 and Thr at position 197.
[19" claim-type="Currently amended] The method of claim 17 or 18,
In the active site cavity, an enzyme molecule characterized in that the amino acid corresponding to Glu at position 46, Glu at position 145 and Cys at position 171 in SEQ ID NO: 1 or 2 is hydrogen-bonded through a water molecule or Its active fragments.
[20" claim-type="Currently amended] The method according to any one of claims 16 to 19,
The DN-carbamoyl-α-amino acid is DN-carbamoyl-phenylglycine, DN-carbamoyl-parahydroxyphenylglycine, DN-carbamoyl-parahydroxyphenylglycine, DN-carba DN-carbamoyl-phenylalanine, DN-carbamoyl-valine, DN-carbamoyl-alanine, DN-carbamoyl-cysteine (DN -carbamoyl-cysteine, DN-carbamoyl-asparatic acid, DN-carbamoyl-glutamic acid, DN-carbamoyl-glycine (DN-carbamoyl- glycine, DN-carbamoyl-histidine, DN-carbamoyl-isoleucine, DN-carbamoyl-lysine, DN DN-carbamoyl-leucine, DN-carbamoyl-methionine, DN-carbamoyl-asparagine, DN-carbamoyl-asparagine DN-carbamoyl-proline, DN-carbamoyl-glutamine, DN-carbamoyl-arginine, DN-carbamoyl-serine DN-carbamoyl-serine, DN-carbamoyl-threonine, DN-carbamoyl-tryptophan and DN-carbamoyl-tyrosine Enzyme molecule or active fragment thereof, characterized in that selected from the group consisting of
[21" claim-type="Currently amended] The method according to claim 14 or 15,
Decarbamylase or mutants or active fragments thereof constructed by the method of molecular design from the conformation of decarbamylase, and the complex conformation of DN-carbamoyl-α-amino acids or D-α-amino acids Characterized decarbamylase complex.
[22" claim-type="Currently amended] A method for designing a decarbamylase mutant, wherein the physical properties and / or functions are modified based on the steric structure of the decarbamylase according to any one of claims 14, 16 and 21. A method comprising designing a modified decarbamylase mutant.
[23" claim-type="Currently amended] A process for producing crystals of enzymes having decarbamylase activity, a process for determining the steric structure of the crystals by X-ray crystallography (X-line crystallization) and the determined conformation A method of designing a decarbamylase mutant, comprising the step of designing a decarbamylase mutant having improved physical properties and / or function based on the structure.
[24" claim-type="Currently amended] The method of claim 23, wherein
23. The method of claim 14, wherein the conformation is a conformation of the decarbamylase according to any one of claims 14, 16 or 21.
[25" claim-type="Currently amended] A process for producing an enzyme crystal having decarbamylase activity, a process for determining the steric structure of the crystal by X-ray crystal structure analysis of the crystal, and improved physical properties and / or functions based on the determined steric structure A method for producing a decarbamylase mutant, comprising the step of designing a decarbamylase mutant and producing the decarbamylase mutant.
[26" claim-type="Currently amended] The method of claim 25,
The steric structure is a steric structure of the decarbamylase according to any one of claims 14, 16 or 21.
[27" claim-type="Currently amended] The method of claim 26,
The process of designing the decarbamylase mutant may include changing the substrate specificity of the enzyme, changing the specific activity of the enzyme, improving the stability of the enzyme, optimizing the optimum pH of the enzyme, and A method for modifying one or more enzyme properties selected from the group consisting of water soluble changes of enzymes.
[28" claim-type="Currently amended] The method of claim 27,
Wherein the alteration of the enzyme properties comprises improving the stability of the enzyme.
[29" claim-type="Currently amended] The method of claim 28,
And wherein the design of the mutant for improving the stability of the enzyme comprises a mutation replacing an amino acid residue which results in a decrease in activity by air oxidation.
[30" claim-type="Currently amended] The method of claim 27,
Wherein said alteration of enzyme properties comprises alteration of enzyme inactivity and optimization of optimal pH of the enzyme.
[31" claim-type="Currently amended] The decarbamylase mutant obtained by the said manufacturing method in any one of Claims 25-30.
[32" claim-type="Currently amended] The steric structure of the decarbamylase crystals according to any one of claims 1 to 7 or the steric structure of the decarbamylase according to any one of claims 14, 16 or 21 is used. Wherein the amino acid primary sequence alters a separate polypeptide or protein enzyme similar to decarbamylase.
[33" claim-type="Currently amended] Means for determining the steric structure of an enzyme crystal having decarbamylase activity by X-ray crystal structure analysis,
Means for designing decarbamylase mutants with improved properties and / or functions based on the determined conformation
System for designing decarbamylase mutant using a computer comprising a.
[34" claim-type="Currently amended] Inputting said crystal data determined by X-ray crystal structure analysis of an enzyme crystal having decarbamylase activity, and
Process for designing decarbamylase mutants with improved properties and / or functions based on determined conformation
A computer-readable recording medium having recorded thereon a program for executing a design process of a decarbamylase mutant comprising a.
[35" claim-type="Currently amended] Inputting said crystal data determined by X-ray crystal structure analysis of an enzyme crystal having decarbamylase activity,
Process for designing decarbamylase mutants with improved properties and / or functions based on determined conformation
A recording medium characterized by recording data describing a three-dimensional structure of a decarbamylase mutant obtained by a treatment comprising a.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Sugio et al.1995|Crystal structure of a D-amino acid aminotransferase: how the protein controls stereoselectivity

---

Wang et al.1985|Bovine chymotrypsinogen A: X-ray crystal structure analysis and refinement of a new crystal form at 1.8 Å resolution

---

Klein et al.1992|Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis

---

Romier et al.1996|Crystal structure of tRNA‐guanine transglycosylase: RNA modification by base exchange.

---

Conti et al.1997|Structural basis for the activation of phenylalanine in the non‐ribosomal biosynthesis of gramicidin S

---

Mulichak et al.1991|Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-. ANG. resolution

---

Sutton et al.1998|Structure of the protein kinase Cβ phospholipid-binding C2 domain complexed with Ca2+

---

Pannifer et al.2001|Crystal structure of the anthrax lethal factor

---

Liao et al.1996|The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr

---

Abad‐Zapatero et al.1996|Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents

---

Luo et al.2001|Crystal structure of LexA: a conformational switch for regulation of self-cleavage

---

Burley et al.1992|Structure determination and refinement of bovine lens leucine aminopeptidase and its complex with bestatin

---

Clausen et al.1996|Crystal Structure of the Pyridoxal-5′-phosphate Dependent Cystathionine β-lyase fromEscherichia coliat 1.83 Å

---

Alexeev et al.1998|The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme

---

DE60126970T2|2007-11-22|Crystal structure of bace and uses thereof

---

Müller et al.1992|Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 Å resolution: A model for a catalytic transition state

---

Lim et al.1997|The crystal structure of an Fe-superoxide dismutase from the hyperthermophile Aquifex pyrophilus at 1.9 Å resolution: structural basis for thermostability

---

Ji et al.1995|Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods

---

Rossjohn et al.1998|A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications

---

Lewis et al.2001|A structural genomics approach to the study of quorum sensing: crystal structures of three LuxS orthologs

---

Yang et al.1998|Identification of the ice-binding surface on a type III antifreeze protein with a “flatness function” algorithm

---

Hennig et al.1995|2.0 Å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

---

Watson et al.1999|Unusual ribulose 1, 5-bisphosphate carboxylase/oxygenase of anoxic Archaea

---

Schrag et al.1993|1· 8 Å refined structure of the lipase from Geotrichum candidum

---

Palm et al.1996|A covalently bound catalytic intermediate in Escherichia coli asparaginase: Crystal structure of a Thr‐89‐Val mutant

同族专利:

---

公开号 | 公开日

---

EP1209234A4|2003-05-02|

---

EP1209234A1|2002-05-29|

---

JP2001069981A|2001-03-21|

---

WO2001016337A1|2001-03-08|

---

CN1376202A|2002-10-23|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
1999-08-31|Priority to JP24679799A

---

1999-08-31|Priority to JPJP-P-1999-00246797

---

2000-08-30|Application filed by 다케다 마사토시, 가네가후치 가가쿠 고교 가부시키가이샤

---

2000-08-30|Priority to PCT/JP2000/005901

---

2002-05-17|Publication of KR20020037044A

优先权:

---

申请号 | 申请日 | 专利标题

---

JP24679799A|JP2001069981A|1999-08-31|1999-08-31|Steric structure of decarbamylase and its utilization|

---

JPJP-P-1999-00246797|1999-08-31|

---

PCT/JP2000/005901|WO2001016337A1|1999-08-31|2000-08-30|Stereostructure of decarbamylase and method of using the same|