PeiW-CD (pseudopeptidoglycan truncated endo-isopeptidase) and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses pseudopeptidoglycan truncated endo-isopeptidase PeiW-CD and application of the pseudopeptidoglycan truncated endo-isopeptidase PeiW-CD, and the amino acid sequence of the pseudopeptidoglycan truncated endo-isopeptidase PeiW-CD is shown as SEQ ID NO: 1; atoms in a crystal three-dimensional structure of the endo-isopeptidase PeiW-CD have at least 40% of atomic coordinates listed in a table 1; or the average root variance of the atomic structure coordinates of the main chain carbon skeleton of at least 40% of amino acids of the crystal three-dimensional structure and the coordinates in the table 1 is smaller than or equal to # imgabs0. The three-dimensional crystal structure of the protein is beneficial to clarification of the catalytic cracking mechanism of the protein, so that the protein has a wide application prospect in the aspect of biotechnology application of methanogenic archaea.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to pseudopeptidoglycan truncated endopeptidase PeiW-CD and its application. Background Art

[0002] Methanogenic archaea have unique methane-producing abilities and play a key role in global carbon cycling, climate change, human health, and energy consumption. However, their unique cell wall structure and drug resistance limit their genetic manipulation and related biotechnological applications. Pseudopeptidoglycan is a key component of the cell wall of methanogenic archaea, which has unique resistance to known bacterial cell wall hydrolases and lysozymes, helping to maintain the rigidity of methanogens and their ability to survive in extreme environments.

[0003] Studies have shown that the pseudopeptidoglycan endoisopeptidase PeiW from the methanogenic archaeal phage (Methanothermobacterphage psiM100) has the ability to cleave archaeal cell walls, and that the pseudopeptidoglycan endoisopeptidase PeiW is only active under divalent metals. The potential cleavage site was inferred using a cell wall analog modified with 4-nitroaniline, but its biochemical characteristics and catalytic mechanism are still unclear, making it impossible to better utilize it for the development of biotechnology.

[0004] Therefore, this application deeply explores the pseudopeptidoglycan truncated endopeptidase PeiW-CD and its applications. By analyzing the PeiW-CD crystal structure, it provides inspiration for understanding the cleavage pattern of this type of enzyme. At the same time, it may have broad application prospects in the development of antibiotics for methanogenic archaea, control and emission of rumen methane, and ecological energy conservation. Summary of the invention

[0005] The purpose of the present application is to provide a method for expressing and purifying a truncated endopeptidase of a methanogenic archaeal pseudopeptidoglycan, its crystal structure and application.

[0006] The present application includes providing a method for genetically engineering the truncated endopeptidase PeiW-CD of the pseudopeptidoglycan derived from a methanogenic archaeal phage to obtain efficient expression, purification and crystallization in Escherichia coli, as well as the three-dimensional crystal structure of the amino acids 1 to 148 of the endopeptidase PeiW-CD and its industrial application. On the one hand, the present application provides a three-dimensional crystal structure of the endopeptidase PeiW-CD and its product, which describes the secondary structure composition, peptide chain orientation, organizational pattern and three-dimensional molecular structure of the endopeptidase PeiW-CD. Among them, diffraction is performed on the endopeptidase PeiW-CD to obtain crystal diffraction data of the endopeptidase PeiW-CD protein, and the structure is analyzed using the model built in the α-fold2 database, and finally the three-dimensional structure of the endopeptidase PeiW-CD protein is obtained, and there are two product molecules glutamate-γ-alanine (EγA) located in the structure, one of which is located near the active center;

[0007] The present application provides a pseudopeptidoglycan truncated endopeptidase PeiW-CD, the amino acid sequence of which is shown in SEQ ID NO: 1; the endopeptidase PeiW-CD contains 147 amino acids and has a molecular weight of about 16.9 KDa; the atoms in the crystal three-dimensional structure of the endopeptidase PeiW-CD have at least 40% of the atomic coordinates listed in Table 1, and / or the mean root variance of the atomic structure coordinates of the main chain carbon skeleton of at least 40% of the amino acids in the crystal three-dimensional structure and the coordinates in Table 1 is less than or equal to

[0008] Furthermore, the resolution of the crystal three-dimensional structure of the endopeptidase PeiW-CD is

[0009] The crystal structure space group of the endopeptidase PeiW-CD is P 121, and one asymmetric unit has only four molecules; the unit cell parameters are: α=γ=90°, β=102.03°;

[0010] Specifically, the crystal structure of the endoisopeptidase PeiW-CD or its foldable part described in the present application has a three-dimensional structure determined by the atomic coordinates (x, y, z)±(1.0, 1.0, 1.0) listed in Table 1.

[0011] Furthermore, the three-dimensional crystal structure is composed of five α-helices and four β-folds, and contains two specific product molecules glutamate-γ-alanine (EγA), one located in the active pocket and the other located on the surface of the structure;

[0012] Among them, α helix 1, i.e., the amino acid segment containing His9-Ile18, α helix 2, i.e., the amino acid segment containing Ala24-Thr34, α helix 3, i.e., the amino acid segment containing Asn47-Thr55, α helix 4, i.e., the amino acid segment containing Cys61-Met75, β fold 1, i.e., the amino acid segment containing Glu78-Cys88, β fold 2, i.e., the amino acid segment containing Gly91-Gly102, β fold 3, i.e., the amino acid segment containing Thr110-Trp112, α helix 5, i.e., the amino acid segment containing Tyr114-Thr120, and β fold 4, i.e., the amino acid segment containing Ala131-Trp138.

[0013] Furthermore, the α helix 1 to the α helix 4 are located in the N-terminal subdomain, and the α helix 5 and the β fold 1 to the β fold 4 are located in the C-terminal subdomain.

[0014] Furthermore, it also includes an enzymatic active center, which is composed of a catalytic triad consisting of cysteine ​​at position 61, histidine at position 96 and aspartic acid at position 113.

[0015] Furthermore, the specific product molecule glutamic acid-γ-alanine (EγA) located in the active pocket and the surrounding amino acid residues, wherein the surrounding amino acid residues include tyrosine at position 41, threonine at position 62 and histidine at position 83, stabilize the conformation through hydrogen bonding. The specific product molecule glutamic acid-γ-alanine (EγA) located on the surface of the structure stabilizes the conformation with glycine at position 109 through hydrogen bonding.

[0016] On the other hand, the present application provides the use of the above-mentioned pseudopeptidoglycan truncated endopeptidase PeiW-CD in catalyzing the hydrolysis of pseudopeptidoglycan isopeptide chains.

[0017] On the other hand, the present application provides the biotechnological application of the above-mentioned endopeptidase PeiW-CD or the above-mentioned application in drug research and development.

[0018] Beneficial effects:

[0019] 1. This application uses in vitro enzyme activity experiments to explore in detail the substrate specificity of the PeiW catalytic domain PeiW-CD.

[0020] 2. The present application uses a crystallization method to obtain a co-crystal of the endoisopeptidase PeiW-CD and its product glutamate-γ-alanine (EγA), and resolves the three-dimensional crystal structure of the endoisopeptidase PeiW-CD.

[0021] 3. The PeiW-CD crystal structure provides inspiration for understanding the cleavage mode of this type of enzyme, and it can also have broad application prospects in the development of antibiotics for methanogenic archaea, control and emission of rumen methane, and ecological energy conservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a polyacrylamide gel electrophoresis analysis of the endopeptidase PeiW-CD (the lanes are represented from left to right: M, protein maker; top, cell disruption supernatant; bottom, cell disruption precipitate; flow, flow-through; 1-9 are protein collection solutions).

[0023] Figure 2 This is a crystal picture of the endopeptidase PeiW-CD.

[0024] Figure 3 The three-dimensional structure ribbon diagram of the endopeptidase PeiW-CD. The α helix is ​​represented by red, the β fold is represented by yellow, the random coil is represented by green, and the product glutamate-γ-alanine (EγA) is represented by a ball-and-stick model. A: monomer c of PeiW-CD; B: isomeric unit of PeiW-CD (four molecules).

[0025] Figure 4 This is the substrate specificity diagram of the endopeptidase PeiW-CD. Among them A: artificially designed substrate peptide, sub 1: γGlu-Lys(Ala-γGlu)-γGlu-Lys(Ala-γGlu); sub 2: γGlu-Lys(Ala-γGlu)-γGlu-Lys(Ala-βAsp); sub3: γGlu-Lys(Ala-γGlu)-γGlu-Lys(Thr-γGlu)(sub 3); sub 4: γGlu-Lys(Ala-γGlu)-γGlu-Lys(Ser-γGlu); sub 5: γGlu-Lys(Thr-γGlu)-γGlu-Lys(Ala-γGlu); sub 6: γGlu-Lys(Ser-γGlu)-γGlu-Lys(Ala-γGlu); B: The ability of endoisopeptidase PeiW-CD to cleave substrates 1 to 6 in A.

[0026] Figure 5 This is the temperature tolerance diagram of endoisopeptidase PeiW-CD.

[0027] Figure 6 The figure is a schematic diagram of the active site of endopeptidase PeiW-CD, which includes cysteine ​​at position 61, histidine at position 96 and aspartic acid at position 113. DETAILED DESCRIPTION

[0028] The following specific examples are used to further illustrate the content of this application.

[0029] The following content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of this application is limited to these descriptions. For ordinary technicians in the technical field to which this application belongs, several simple deductions or substitutions can be made without departing from the concept of this application, which should be deemed to fall within the scope of protection of this application.

[0030] Example 1. Expression and purification of endopeptidase PeiW-CD in Escherichia coli

[0031] The pseudopeptidoglycan truncated endopeptidase PeiW-CD derived from a methanogenic archaeal phage, the gene sequence of which is suitable for expression in Escherichia coli after codon optimization is shown in SEQ ID NO:2.

[0032] According to the correspondence between codons and amino acids, the amino acid sequence encoding PeiW-CD is shown in SEQ ID NO: 1.

[0033] The gene of endopeptidase PeiW-CD was cloned into pET28a vector by homologous recombination technology to express fusion protein with 6 His fused to the amino terminal.

[0034] The primers for amplifying PeiW-CD are:

[0035] The upstream primer is: catcatcacagcagcATGCAGCCACAGCCGAGCCTGAA

[0036] PeiW-CD_F(5'-3')

[0037] The downstream primer is: gtggtggtggtggtgctcgagTCATTTATCATACACCGGGCCCA

[0038] PeiW-CD_R(5'-3')

[0039] ATG in the upstream primer is the start codon, and TCA in the downstream primer is the stop codon. The lowercase letters are the homology arm sequences upstream and downstream of the multiple cloning site of the pET28a vector. The cloning kit is the ClonExpress UltraOne Step Cloning Kit (C115) of Vazyme, and the cloning steps are completed according to its instructions.

[0040] The recombinant vector was transformed into Escherichia coli BL21 (DE3) and cultured at 37°C with shaking until OD 600When the concentration reached 0.6-0.8, IPTG was added to induce expression at a final concentration of 0.1 mM. The expression conditions were: shaking culture at 16°C for 12 h. The cells were then collected by centrifugation for subsequent purification.

[0041] The expression bacteria collected by centrifugation were suspended in an appropriate amount of buffer [2mM DTT; 5mM CaCl2; 10mM MgCl2; 10mM imidazole; 50mM Tris; 300mM NaCl; 1% Triton X-100 (v / v); 20% glycerol (v / v), pH 7.5], and the bacteria were lysed using a low-temperature ultra-high pressure cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.), and the precipitate and other granular impurities were removed by high-speed centrifugation at 18000rpm for 30min. After the supernatant after centrifugation was combined with the Ni-NTA affinity medium, the medium was washed with a buffer of 1mM DTT; 50mM Tris; 100mM imidazole; 500mM NaCl, pH 7.5 to remove impurities. Finally, the target protein was eluted from the affinity medium with 50 mM Tris, 750 mM imidazole, 500 mM NaCl, pH 7.5, and the eluate was concentrated with 3 KDa and 10 KDa cut-off concentrators ( Figure 1 The concentrated protein solution was further purified by gel filtration chromatography (SuperdexTM 75, 10 / 300GL) using a buffer of 1 mM DTT, 20 mM Mops, 0.2 M NaCl and 10% glycerol (v / v), pH 7.0. The eluted target protein was concentrated to a concentration of 10-15 mg / mL for crystallization experiments, and the remaining sample was stored at -80°C.

[0042] Example 2. Crystallization of endoisopeptidase PeiW-CD

[0043] The endopeptidase PeiW-CD expressed and purified as described in Example 1 was concentrated to a concentration of about 10-15 mg / mL, and a crystallization kit (Crystal Screen Kit I / II, Index, Salt, PEG / Ion, etc. from Hampton Research, etc.) was used as the initial screening conditions for crystal growth, and the sitting drop method was used for crystallization. Figure 2 As shown, the present application obtained initial crystals under multiple different crystallization reagent conditions. Through subsequent optimization and adjustment, 200mM Lithium sulfate; 100mM TRIS pH 8.5; 40.0% v / v Polyethylene glycol 400 buffer was selected as the crystal growth condition, and a set of crystals with a resolution of X-ray diffraction data.

[0044] Example 3. Data collection and structural analysis of endoisopeptidase PeiW-CD

[0045] The prepared crystals in the present application were treated with 15% glycerol as an antifreeze and then frozen in liquid nitrogen. The data collection of crystal X-ray diffraction was carried out at the Shanghai Light Source (SSRF) Biomacromolecule Crystallography Beamline Station (Line 5, Station 6, BL19U). HKL3000 was used for data processing, and the structural analysis used the model built by α-fold2 itself as a reference. The molecular replacement method was used, and the Refmac program was used with the assistance of Coot software for further correction. The crystal structure of the endopeptidase PeiW-CD protein was finally obtained. A person of ordinary skill in the art would know that the atoms in the three-dimensional crystal structure have at least 40% of the atomic coordinates listed in Table 1, or the atomic structure coordinates of the main chain carbon skeleton of at least 40% of the amino acids in the three-dimensional crystal structure of the 3D protein have a mean root mean square deviation of the coordinates in Table 1 that is less than or equal to. The atomic coordinates of the 3D protein can be considered to have the same structure as the 3D protein.

[0046] Specifically, the three-dimensional crystal structure of the endopeptidase PeiW-CD has four protein molecules in one crystallographic asymmetric unit. Figure 3 As shown, the endopeptidase PeiW-CD is composed of 4 β folds and 5 α helices, wherein the α helices 1 to 4 are located in the N-terminal subdomain, and the α helices 5 and β folds 1 to 4 are located in the C-terminal subdomain. Among them, α helix 1, i.e., the amino acid segment containing His9-Ile18, α helix 2, i.e., the amino acid segment containing Ala24-Thr34, α helix 3, i.e., the amino acid segment containing Asn47-Thr55, α helix 4, i.e., the amino acid segment containing Cys61-Met77, β fold 1, i.e., the amino acid segment containing Glu78-Cys88, β fold 2, i.e., the amino acid segment containing Gly91-Gly102, β fold 3, i.e., the amino acid segment containing Thr110-Trp112, α helix 5, i.e., the amino acid segment containing Tyr114-Thr120, and β fold 4, i.e., the amino acid segment containing Ala131-Trp138.

[0047] The atomic coordinates of the PeiW-CD crystal have been uploaded to the RCSB Protein Data Bank (RCSBPDB) database, and the relevant coordinates can be queried by protein number (PDB ID: 8JX4).

[0048] Crystal space group: P 121

[0049] The unit cell parameters are: α=γ=90°, β=102.03°

[0050] I / σ(I):1.66

[0051] Rwork / Rfree are: 0.226 and 0.284 respectively

[0052] Resolution range 35.48-2.30

[0053] Data integrity (%): 98.1

[0054] Table 1

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Example 4. Analysis of the isopeptide chain hydrolysis ability of endoisopeptidase PeiW-CD

[0084] The analysis of the hydrolysis ability of endoisopeptidase PeiW-CD on pseudopeptidoglycan isopeptide chains of methanogenic archaea was carried out using the following system: 0.01 mM pure enzyme protein sample, 0.1 M artificially synthesized pseudopeptidoglycan isopeptide chain substrates 1-6, total reaction volume 500 μL, incubated at 60°C for 30 min, and then the hydrolysis products were detected by LC-MS. In the LC-MS system, solvent A contained 0.05% trifluoroacetic acid (TFA), 2% acetonitrile and water, and solvent B contained 0.05% TFA, 90% acetonitrile and water. The procedure was: 10% solvent A 0 min, 50% solvent B 3 min, 90% solvent B 5-6 min, 5% solvent B 6.1 min. The injection volume was 20 μL, the flow rate was 1 mL / min, and the total time was 8 min.

[0085] like Figure 4 As shown, the preferred substrate side chain of endopeptidase PeiW-CD is glutamic acid-γ-alanine-ε-lysine (EγAεK), wherein the concentrations of dipeptide product EγA after cleavage of substrates 1 to 6 are 187.18±6.26, 70.45±3.01, 35.42±3.25, 44.04±0.67, 53.79±1.62 and 63.05±2.80 μmol L -1 The second is the dipeptide product EγS with a side chain of glutamic acid-γ-serine-ε-lysine (EγSεK). The concentrations of the dipeptide product EγS after cleavage of substrates 4 and 6 were 12.65±0.20 and 16.45±0.40μmolL -1 .

[0086] Example 5. Analysis of temperature tolerance of endoisopeptidase PeiW-CD

[0087] The specific operation is as follows: 50 mM Bistris propane buffer (pH 7.85), 10 mM CaCl2, 100 μM substrate Glu-γ-Ala-pNA (EγApNA), add 15 μM pure enzyme protein sample, and continuously measure the absorbance value A at 30, 40, 50, 60, 70 and 80 °C. 385 10 min. Figure 5 As shown, the temperature tolerance of endoisopeptidase PeiW-CD was measured in the range of 30 to 80° C. The results showed that PeiW-CD had good enzyme activity at 30 to 70° C.

[0088] The above is only a preferred embodiment of the present application. It should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge in the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present application, and shall be within the scope of protection of the claims attached to the present application.

Claims

1. Pseudopeptidoglycan truncated endopeptidase PeiW-CD, characterized in that The amino acid sequence of the pseudopeptidoglycan truncated endopeptidase PeiW-CD is shown in SEQ ID NO: 1; the atoms in the crystal three-dimensional structure of the endopeptidase PeiW-CD have at least 40% of the atomic coordinates listed in Table 1; and / or, the mean root square deviation of the atomic structure coordinates of the main chain carbon skeleton of at least 40% of the amino acids in the crystal three-dimensional structure and the coordinates in Table 1 is less than or equal to 2. The endopeptidase PeiW-CD according to claim 1, characterized in that The resolution of the crystal three-dimensional structure of the endopeptidase PeiW-CD is The crystal structure space group of the endopeptidase PeiW-CD is P121, and one asymmetric unit has only four molecules; the unit cell parameters are: α=γ=90°, β=102.03°.

3. The endopeptidase PeiW-CD according to claim 1, characterized in that: The three-dimensional crystal structure consists of five α-helices and four β-folds, and contains two specific product molecules, glutamate-γ-alanine (EγA), one located in the active pocket and the other located on the surface of the structure; Among them, α helix 1, i.e., the amino acid segment containing His9-Ile18, α helix 2, i.e., the amino acid segment containing Ala24-Thr34, α helix 3, i.e., the amino acid segment containing Asn47-Thr55, α helix 4, i.e., the amino acid segment containing Cys61-Met75, β fold 1, i.e., the amino acid segment containing Glu78-Cys88, β fold 2, i.e., the amino acid segment containing Gly91-Gly102, β fold 3, i.e., the amino acid segment containing Thr110-Trp112, α helix 5, i.e., the amino acid segment containing Tyr114-Thr120, and β fold 4, i.e., the amino acid segment containing Ala131-Trp138.

4. The endopeptidase PeiW-CD according to claim 3, characterized in that: The α helix 1 to the α helix 4 are located in the N-terminal subdomain, and the α helix 5 and the β fold 1 to the β fold 4 are located in the C-terminal subdomain.

5. The crystal three-dimensional structure of endopeptidase PeiW-CD according to claim 4, characterized in that: The invention also comprises an enzymatic active center, wherein the enzymatic active center is composed of a catalytic triad consisting of cysteine ​​at position 61, histidine at position 96 and aspartic acid at position 113.

6. The endopeptidase PeiW-CD according to claim 5, characterized in that: It also includes a specific product molecule glutamic acid-γ-alanine (EγA) located in the active pocket and surrounding amino acid residues, wherein the surrounding amino acid residues include tyrosine at position 41, threonine at position 62 and histidine at position 83, which stabilize the conformation through hydrogen bonding. The specific product molecule glutamic acid-γ-alanine (EγA) located on the surface of the structure stabilizes the conformation with glycine at position 109 through hydrogen bonding.

7. Use of the pseudopeptidoglycan truncating endopeptidase PeiW-CD according to any one of claims 1 to 6 in catalyzing the hydrolysis of pseudopeptidoglycan isopeptide chains.

8. The use according to claim 7, characterized in that: The temperature for catalyzing the hydrolysis of pseudopeptidoglycan isopeptide chains is 30-70°C.

9. The endopeptidase PeiW-CD according to any one of claims 1 to 6 or the use according to any one of claims 7 to 8, used in biotechnology for drug development.

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