Hypoxanthine-guanine phosphoribosyltransferase mutant and its application

Abstract

The present invention provides a hypoxanthine-guanine phosphoribosyltransferase mutant and its application. The mutant is obtained by the following single mutations at positions 124, 154, or 182 of the wild-type hypoxanthine-guanine phosphoribosyltransferase amino acid sequence: V124D / E / L / F / Y, K154R / W, D182Y, where " / " represents "or". The present invention screened and obtained a hypoxanthine-guanine phosphoribosyltransferase mutant with a selective catalytic reduction activity significantly higher than that of the wild-type. The hypoxanthine-guanine phosphoribosyltransferase is used as a biocatalyst for the synthesis of hypoxanthine and PRPP, and has the advantages of high heterologous expression efficiency, good thermal stability, simple reaction steps, and mild reaction conditions. The present invention discovered a mutant with significantly better catalytic performance than the wild-type, enhancing the application potential of the reverse reaction of hypoxanthine-guanine phosphoribosyltransferase.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a hypoxanthine-guanine phosphoribosyltransferase mutant and its application, in particular to the application of the hypoxanthine-guanine phosphoribosyltransferase mutant in the generation of purine compounds and 5-phosphoribosyl-1-pyrophosphate. Background Art

[0002] Hypoxanthine (Hx), a common base in living organisms, is an important intermediate for antiviral drugs and drugs related to nucleic acid metabolism. Phosphoribosyl pyrophosphate (PRPP) participates in multiple metabolic pathways in the human body, including DNA / RNA synthesis, histidine synthesis, and tryptophan synthesis. As a key biosynthetic component, PRPP can be used to produce biomolecules related to DNA, RNA, and other nucleotides, and is used to synthesize drugs related to nucleic acid metabolism. Industrially, hypoxanthine is primarily obtained through chemical synthesis, which involves the introduction of oxygen groups into specific sites on purines. Although hypoxanthine is currently relatively cheap, it requires high-temperature and high-pressure reaction conditions. PRPP, on the other hand, is unstable, difficult to synthesize industrially, and extremely expensive. Therefore, there is an urgent need to develop efficient and environmentally friendly methods for the synthesis of hypoxanthine and PRPP.

[0003] Compared with chemical catalysis, the use of phosphoribosyltransferase to catalyze the synthesis of hypoxanthine or PRPP has mild reaction conditions, is easy to control, and is environmentally friendly. However, wild-type enzymes usually have low catalytic efficiency and poor thermal stability. Therefore, improving the catalytic performance of wild-type enzymes through mutation and developing phosphoribosyltransferases with industrial application value are of great significance for drug synthesis and structural modification.

[0004] Hypoxanthine-guaninephosphoribosyltransferase (HGPRT) belongs to the 6-oxopurine phosphoribosyltransferase (PRTase) family. Its currently reported biological function is to catalyze the condensation of guanine / hypoxanthine with 5-phosphoribosyl-1-pyrophosphate (PRPP) to form guanine / inosine monophosphate (GMP / IMP). It is a key enzyme in the salvage synthesis pathway of guanine and hypoxanthine. At the same time, HGPRT catalyzes the reverse process of the above reaction, using guanine / inosine monophosphate (GMP / IMP) and pyrophosphate as substrates and Mg as the substrate. 2+HGPRT is a catalytically active molecule that uses 6-oxopurine (GUA / Hx) as a cofactor to generate 5-phosphoribosyl-1-pyrophosphate (PRPP). HGPRT has high heterologous expression efficiency and good thermal stability, suggesting promising applications. However, its catalytic efficiency is relatively low, necessitating further screening for highly efficient mutants with potential for industrial application. Summary of the Invention

[0005] The present invention aims to provide a hypoxanthine-guanine phosphoribosyltransferase mutant, aiming to improve the activity of the reverse reaction and realize its industrial application. The mutant is obtained by incorporating the following single mutations at positions 124, 154, and 182 of the wild-type hypoxanthine-guanine phosphoribosyltransferase amino acid sequence: V124D / E / L / F / Y, K154R / W, and D182Y, where " / " represents "or." Specifically, the mutant is based on the wild-type hypoxanthine-guanine phosphoribosyltransferase amino acid sequence SEQ ID NO. 10 (its DNA sequence is SEQ ID NO. 1) and undergoes one of the following mutations: V124D, V124E, V124L, V124F, V124Y, K154R, K154W, and D182Y.

[0006] Any hypoxanthine-guanine phosphoribosyltransferase mutant comprising the above mutations, which has deleted, inserted or substituted one or more amino acids and still has the activity of the above hypoxanthine-guanine phosphoribosyltransferase mutant, still falls within the scope of protection of the present invention.

[0007] Another object of the present invention is to provide a recombinant vector comprising the gene encoding the hypoxanthine-guanine phosphoribosyltransferase mutant.

[0008] Another object of the present invention is to provide the use of the hypoxanthine-guanine phosphoribosyltransferase mutant as a biocatalyst in the synthesis of hypoxanthine and PRPP. That is, the application in improving the degree of reverse reaction and synthesizing hypoxanthine and 5-phosphoribosyl-1-pyrophosphate (PRPP). Specifically, in the reaction, the hypoxanthine-guanine phosphoribosyltransferase mutant catalyzes the condensation of IMP and pyrophosphate to generate hypoxanthine (Hx) and PRPP. The synthesis reaction uses IMP and sodium pyrophosphate (DP) as raw materials, Mg 2+ As a cofactor, hypoxanthine and PRPP are generated by the catalysis of hypoxanthine-guanine phosphoribosyltransferase mutant, as shown in reaction formula 1:

[0009]

[0010] Furthermore, the present invention provides a method for synthesizing hypoxanthine and PRPP, using Mg 2+As a cofactor, IMP and sodium pyrophosphate undergo a condensation reaction in a buffer solution under the catalysis of a hypoxanthine-guanine phosphoribosyltransferase mutant to generate hypoxanthine and PRPP. The amount of IMP added is 0.5-30 mM, the amount of sodium pyrophosphate added is 0.1-30 mM, the amount of MgCl2 added is 0.15-45 mM, the amount of the hypoxanthine-guanine phosphoribosyltransferase mutant added is 0.1-5 mg / mL, the pH value is 5-10, the reaction temperature is 15-45°C, and the reaction time is 0.5-24 h.

[0011] The reaction system of the present invention comprises a hypoxanthine-guanine phosphoribosyltransferase mutant, IMP, sodium pyrophosphate substrate and MgCl2, and can catalyze the generation of hypoxanthine and PRPP under the conditions of controlled pH and temperature.

[0012] Specifically, hypoxanthine-guanine phosphoribosyltransferase is derived from Mycobacterium tuberculosis. The nucleotide sequence encoding the hypoxanthine-guanine phosphoribosyltransferase is derived from GenBank and is numbered U88876.1. After codon optimization, it is shown as MtHGPRT-DNA (SEQ ID No. 1) in the sequence listing.

[0013] Specifically, the amino acid sequence of hypoxanthine-guanine phosphoribosyltransferase is shown as MtHGPRT-AA (SEQ ID No. 10) in the sequence listing.

[0014] As known to those skilled in the art, the nucleotide sequence of the hypoxanthine-guanine phosphoribosyltransferase gene of the present invention may also be any other nucleotide sequence encoding the amino acid sequence shown in MtHGPRT-AA (SEQ ID No. 10) in the sequence listing.

[0015] Any nucleotide sequence obtained by substituting, modifying or inserting one or more nucleotides into the nucleotide sequence shown in MtHGPRT-DNA, as long as it has more than 90% homology with the nucleotide sequence, falls within the scope of protection of the present invention.

[0016] Any deletion, insertion or substitution of one or more amino acids in the amino acid sequence of MtHGPRT-AA that has the activity of generating PRPP and hypoxanthine still falls within the scope of protection of the present invention.

[0017] The present invention has the following beneficial effects: through rational mutagenesis based on the three-dimensional structure of hypoxanthine-guanine phosphoribosyltransferase, mutants with significantly enhanced reverse reaction activity are obtained. The enzyme activities of the mutants V124F, V124Y, K154R, and D182Y are 5.81 times, 5.45 times, 2.59 times, and 3.56 times that of the wild-type enzyme, respectively. The present invention has certain significance for the industrial application of hypoxanthine-guanine phosphoribosyltransferase, increasing the enzyme's potential for application in the pharmaceutical and chemical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Blank control HPLC graph: The reaction solution without enzyme addition is used as the blank, and only the substrate IMP is added.

[0019] Figure 2 Mg 2+ High performance liquid chromatogram of the reaction catalyzed by hypoxanthine-guanine phosphoribosyltransferase and its mutants as cofactor, where A. wild type MtHGPRT, B. MtHGPRT-V124F, C. MtHGPRT-V124Y. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0021] The experimental methods in the present invention are all conventional methods unless otherwise specified.

[0022] The plasmid extraction kit in the present invention was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; E. coli DH5α, E. coli BL21 (DE3) and the like were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; and the prestained protein marker was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.

[0023] Common reagents used in the present invention, including substrates, were purchased from Aladdin Chemical Reagent Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., and Sinopharm Chemical Reagent Co., Ltd.

[0024] Example 1 Expression of Hypoxanthine-Guanine Phosphoribosyltransferase

[0025] The sequence of the gene encoding Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (GenBank: U88876.1) was codon-optimized (sequence as SEQ ID NO. 1) and fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pET-28a(+) vector to construct the MtHGPRT-pET-28a(+) plasmid. After sequence verification, the plasmid was heat-shock transformed into E. coli BL21(DE3) competent cells to obtain hypoxanthine-guanine phosphoribosyltransferase-expressing engineered bacteria. A single colony was picked from LB plate medium containing 50 μg / ml kanamycin and inoculated into LB liquid medium containing 50 μg / ml kanamycin. The culture was shaken at 37°C and 200 rpm for 12 h, then transferred to 3 L liquid LB medium for expansion culture and continued to shake at 37°C and 200 rpm for 8 h. When the optical density OD 600 When the pH value reached 0.6, the temperature was lowered to 16°C, and IPTG solution with a final concentration of 0.7 mM was added to induce expression for 16 h. The culture solution was centrifuged at 8000 rpm for 10 min, the supernatant culture solution was discarded, and the bacteria were stored at -20°C for later use.

[0026] Example 2 Purification of Hypoxanthine-Guanine Phosphoribosyltransferase

[0027] Resuspend 3 g of engineered bacteria expressing hypoxanthine-guanine phosphoribosyltransferase in 20 ml of lysis buffer (10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20, pH 8.0). Shake thoroughly and add lysozyme (1 mg / ml). Incubate on ice for 40 minutes. Ultrasonicate for three 3-minute cycles, with 15-minute intervals between each cycle. Centrifuge at 14,000 rpm for 15 minutes. The resulting supernatant is the crude enzyme solution. Ni-IDA protein purification magnetic beads were used as the purification material, and the single-use volume was 5 ml of 10% magnetic bead suspension. 10 ml of lysis buffer was used to equilibrate the Ni-IDA magnetic beads. After magnetic separation, the crude enzyme solution was added, and the mixture was mixed and incubated at 4°C for 1 hour. The unadsorbed protein was eluted with lysis buffer (50 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20 pH 8.0). Finally, the target protein was eluted and collected with elution buffer (500 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20 pH 8.0). The target protein was dialyzed with 5 L of Kpi buffer (50 mM KH2PO4, 50 mM K2HPO4, pH 7.4) to remove salt and imidazole.

[0028] Example 2 Preparation of Hypoxanthine-Guanine Phosphoribosyltransferase Mutants

[0029] Through structural and amino acid sequence comparison, valine 124, aspartic acid 182 and lysine 154 of hypoxanthine-guanine phosphoribosyltransferase were identified as key substrate binding sites. Using the plasmid MtHGPRT-pET-28a(+) containing wild-type hypoxanthine-guanine phosphoribosyltransferase DNA as a template, site-directed mutagenesis was performed on the above three sites.

[0030] Table 1. Primers used for hypoxanthine-guanine phosphoribosyltransferase mutation

[0031]

[0032] After verification by sequencing, each mutant product was transformed into E. coli BL21 competent cells for expression to obtain hypoxanthine-guanine phosphoribosyltransferase mutant expression engineered bacteria.

[0033] The expression and purification methods of the hypoxanthine-guanine phosphoribosyltransferase mutant were the same as those of the wild type.

[0034] Example 3 Synthesis of hypoxanthine and PRPP by wild-type and mutant hypoxanthine-guanine phosphoribosyltransferase

[0035] The hypoxanthine-guanine phosphoribosyltransferase or mutant obtained in Example 2 was added to the reaction system at a concentration of 1.0 mg / ml, and 50 mM Kpi (pH 7.4) was used as a buffer. IMP, 0.8 mM sodium pyrophosphate, and 10 mM MgCl2 were added to a final concentration of 0.8 mM, respectively. The mixture was shaken at 37°C (666 rpm) for 4.0 h, and then an equal volume of methanol was added to terminate the reaction. The reaction solution was centrifuged at 14,000 rpm for 15 min, and the supernatant was sampled and analyzed by high-performance liquid chromatography (HPLC) to determine the amount of substrate and product. The HPLC analysis method was as follows: chromatograph: Agilent HPLC 1260; column: Extend-C18 4.6*250 mm; column temperature: 30°C; flow rate: 0.7 ml / min; detection wavelength: 245 nm; mobile phase: 80% water, 20% methanol. The conversion rate and yield of Hx catalyzed by hypoxanthine-guanine phosphoribosyltransferase and its mutants were calculated based on the concentration curve of IMP and Hx standards, and the relative activities of the mutants were compared.

[0036] Table 2. Hypoxanthine-guanine phosphoribosyltransferase activity assay

[0037]

[0038] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A hypoxanthine-guanine phosphoribosyltransferase mutant, characterized in that: The amino acid sequence of the mutant is based on SEQ ID NO. 10, in which the valine at position V124 is mutated to phenylalanine: V124F.

2. A coding gene, characterized in that Encoding the hypoxanthine-guanine phosphoribosyltransferase mutant according to claim 1.

3. A recombinant vector, characterized in that Contains the coding gene according to claim 2.

4. Use of the hypoxanthine-guanine phosphoribosyltransferase mutant according to claim 1 as a biocatalyst in the reaction of synthesizing hypoxanthine and PRPP.

5. The use according to claim 4, characterized in that The synthesis of hypoxanthine and PRPP is achieved by the following steps: IMP and pyrophosphate are used as raw materials, Mg 2+ As a cofactor, the hypoxanthine-guanine phosphoribosyltransferase mutant according to claim 1 is used as a biocatalyst to carry out a condensation reaction in a buffer solution. The reaction formula is as follows: 。 6. The use according to claim 5, characterized in that In the hypoxanthine-guanine phosphoribosyltransferase catalyzed reaction, the amount of IMP added is 0.5-30 mM, the amount of sodium pyrophosphate added is 0.1-30 mM, the amount of MgCl2 added is 0.15-45 mM, the amount of hypoxanthine-guanine phosphoribosyltransferase mutant added is 0.1-5 mg / mL, the pH value is 5-10, the reaction temperature is 15-45°C, and the reaction time is 0.5-24 h.

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