A nicotinamide riboside kinase mutant, its design method and application

Abstract

The present invention relates to a nicotinamide ribokinase mutant, its design method and application. The mutant is obtained by mutating the 172nd amino acid residue in the amino acid sequence of nicotinamide ribokinase from valine to lysine. The amino acid sequence of the nicotinamide ribokinase is as shown in SEQ ID NO: 2. The BEANRK mutant constructed in this invention has successfully increased the enzyme activity by 1.2 times compared with the wild BEANRK. In a single-enzyme catalysis system without using expensive phosphoribosyl pyrophosphate, the conversion rate of NR to the product NMN can reach 91%. Moreover, the thermal stability of this mutant has also been improved, providing an effective way to improve the performance of nicotinamide ribokinase, reducing production costs, and having broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme structure modification and biosynthesis, and particularly relates to a nicotinamide riboside kinase mutant and a design method and application thereof. Background Art

[0002] β-Nicotinamide mononucleotide (NMN) is a natural bioactive nucleotide. As early as 1948, when Arthur Kornberg studied the components that produce NAD + in yeast cells, it was found that NMN can react with adenosine triphosphate (ATP) to generate NAD + , so NMN is an important precursor for the synthesis of NAD + in the human body. NMN is a product naturally formed by the reaction of a phosphate group with a nucleoside containing ribose and nicotinamide, so NMN also belongs to the vitamin B3 derivative. The pharmacological activity of NMN is mainly mediated by NAD + biosynthesis, and it can improve cardiovascular and cerebrovascular diseases, Alzheimer's disease, type II diabetes caused by NAD + deficiency, as well as obesity caused by age and diet. In many clinical studies, taking NMN can improve the mitochondrial function of different metabolic organs in the body. More and more evidence shows that NMN has broad health care applications and drug treatment values.

[0003] At present, the main production methods of NMN are chemical synthesis method and bioenzyme method. The chemical synthesis method mainly uses nicotinamide as the starting material, which is protected by trimethylsilyl and then catalytically condensed with tetraacetylribose to obtain triacetyl nicotinamide riboside trifluoromethanesulfonate, followed by ion exchange to obtain triacetyl nicotinamide riboside chloride, then alkaline hydrolysis and crystallization to obtain nicotinamide riboside chloride, and finally phosphorylation to obtain NMN. This route involves multiple organic reagents, which are harmful to the environment, and the conditions are delicate and strict, and are not easy to control. In contrast, the bioenzyme method has high substrate specificity, high catalytic efficiency, mild reaction conditions, easy control and environmental friendliness. Therefore, the bioenzyme method is widely used in the production of NMN.

[0004] The production processes by biocatalysis mainly fall into two categories: one is centered around nicotinamide phosphoribosyltransferase, which includes single-enzyme and multi-enzyme catalysis. Since phosphoribosyl pyrophosphate involved in single-enzyme catalysis is extremely expensive, and in multi-enzyme catalysis, there are many substrates and purification is difficult, it is not suitable for industrial production. The other category uses nicotinamide riboside (NR) and ATP as substrate raw materials, and under the catalytic action of nicotinamide riboside kinase (NRK), NMN is obtained through reaction. This method has advantages such as high yield and high product purity, and has become a highly potential method for NMN production. However, currently, the types of NRK that have been explored and applied are very few, and the performance of the enzyme needs to be improved. CN115948365A discloses the application of a mutant of nicotinamide riboside kinase from Saccharomyces cerevisiae and its encoding gene in the production of nicotinamide mononucleotide, and its NMN conversion rate reaches over 70%. Under the condition of a single NRK, the consumption of ATP is large, the conversion rate is low, and the production cost is high. Therefore, in order to achieve industrial-scale NMN production, highly efficient and stable catalytic enzymes still need to be urgently explored and studied. Summary of the Invention

[0005] The present invention provides a mutant obtained by modifying wild-type nicotinamide riboside kinase using techniques such as semi-rational design and amino acid mutation. Compared with the wild-type nicotinamide riboside kinase, the enzyme performance of the mutant has been improved. Applying it to NMN production can achieve the purpose of reducing production costs and increasing the NMN yield, which is helpful for industrial production.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A nicotinamide riboside kinase mutant, wherein the mutant is obtained by mutating the 172nd amino acid residue in the amino acid sequence of nicotinamide riboside kinase from valine to lysine;

[0008] The amino acid sequence of the nicotinamide riboside kinase is as shown in SEQ ID NO: 2. SEQ ID NO: 1 is its nucleotide sequence.

[0009] Another object of the present invention is to provide a design method for the above mutant, including:

[0010] According to the size of the active pocket and the distribution of the substrate channel of nicotinamide riboside kinase, select nicotinamide riboside kinase with a relatively large active pocket and a long and narrow substrate channel distribution as the mutant base enzyme;

[0011] Perform homology modeling on the mutant base enzyme to obtain its three-dimensional structure model;

[0012] Molecular docking was performed on the tertiary structure model of the mutant base enzyme with the substrates nicotinamide ribose and adenosine triphosphate, and amino acids within the docking sites of the substrate and the enzyme and the Lid structure were selected as alternative mutation sites to design single mutants and combined mutants; Single mutants and combined mutants were designed with amino acids within the docking sites of the substrate and the enzyme and the Lid structure as alternative mutation sites;

[0013] Mutants were constructed for catalytic reactions, and optimized mutants were selected based on enzyme activity and thermal stability.

[0014] As a preferred embodiment, after homology modeling, the Ramachandran plot of the drawn model was used to evaluate the rationality of the modeling results.

[0015] Another object of the present invention is to provide the application of the above-mentioned mutants in the synthesis of β-nicotinamide mononucleotide.

[0016] As a preferred embodiment, the mutant synthesizes β-nicotinamide mononucleotide in a single-enzyme catalytic system.

[0017] As a preferred embodiment, the mutant synthesizes β-nicotinamide mononucleotide using nicotinamide ribose as a substrate.

[0018] As a preferred embodiment, the reaction system for the mutant to synthesize β-nicotinamide mononucleotide includes nicotinamide ribose, adenosine triphosphate, magnesium sulfate heptahydrate, and the pure enzyme solution of the mutant.

[0019] As a preferred embodiment, the dosage of the pure enzyme solution of the mutant in the reaction system is a final protein concentration of 0.1 - 0.5 g / L.

[0020] As a preferred embodiment, the dosage of the substrate in the reaction system is 0.1 - 10 g / L.

[0021] As a preferred embodiment, the mutant synthesizes β-nicotinamide mononucleotide at 20 - 30 °C.

[0022] The beneficial effects of the present invention:

[0023] (1) Based on the structural analysis of the existing commonly used nicotinamide ribokinase database, the present invention found that three NRKs reported and used in patents: HNRK, derived from Human; KLUNRK, derived from Kluyveromyces marxianus; KPNRK, derived from Komagataella phaffii, all have relatively large active pockets near the P-loop motif, and the substrate channels are also distributed in the active pockets. According to this structural feature, among the preliminary predicted structures of 57 heterologous NRKs, a novel NRK enzyme source with a relatively large active pocket and a narrow and long substrate channel distribution was selected. The nicotinamide ribokinase gene BEANRK derived from Beauveria bassiana was determined as the research object. Through the modification of its structure, the optimal mutant M1 is a single-point mutation of the amino acid at position 172. The mutation point V172K changes the original valine to lysine, successfully increasing the enzyme activity of BEANRK by 1.2 times, and also improving the enzyme stability to a certain extent at 20 °C and 30 °C.

[0024] (2) In the single-enzyme catalysis system of the mutant of the present invention without using expensive phosphoribosyl pyrophosphate, the conversion rate of the substrate NR can reach 91%, reducing the industrial production cost.

[0025] (3) The present invention provides a rational structural modification idea, which can provide reference for the further exploration of similar enzymes in the future. The specific method mainly is that first, it is found that the conserved sequence P-loop motif in nicotinamide ribokinase determines the catalytic activity of the enzyme. By selecting based on the size of the active pocket and substrate channel of the P-loop motif in the enzyme structure, it has universality.

[0026] (4) In the present invention, the amino acids within the docking site of the substrate and the enzyme and the Lid structure were selected as alternative mutation points. Then, through sequence analysis, amino acids were replaced with KLUNRK and HNRK as certain reference templates to obtain the optimal mutant M1. Molecular docking shows that the terminal phosphate group of ATP in M1 is closer to the substrate NR, and the shortening of the spatial distance is very beneficial to the transfer of the phosphate group between substrates. Therefore, through sequence alignment and structural information analysis, borrowing the site amino acids of excellent enzymes to improve the enzyme performance is an effective way to modify new enzymes. Brief Description of the Drawings

[0027] Figure 1 It is the docking model of 2qt0 and its comparison with the tertiary structure of BEANRK; A: The structural model 2qt0 of HNRK in the PDB database; B: The structural model of BEANRK.

[0028] Figure 2It is the result of the Laplace diagram analysis of the BEANRK model.

[0029] Figure 3 It is the amino acid alignment result of BEANRK, KLUNRK, and HNRK.

[0030] Figure 4 It is the comparison of the relative enzyme activity and thermal stability between the single-point mutants and WT.

[0031] Figure 5 It is the comparison of the relative enzyme activity and thermal stability between the combined mutants and WT, V172K.

[0032] Figure 6 It is the analysis of the interaction between BEANRK (A) and M1 (B) and the substrate, as well as the difference in the interaction. Detailed implementation mode

[0033] In the examples, the enzyme reaction system used to evaluate the catalytic performance of the mutants is as follows:

[0034] The substrate is 5 g / L NR, 12.5 mM magnesium sulfate heptahydrate, 28 mM ATP. Add the pure enzyme solution with a final protein concentration of 0.2 g / L, and react at 37 °C for 5 min at 900 rpm.

[0035] Perform liquid chromatography analysis on the reaction products using Dionex U-3000. The HC-C18 column is used for determination. The mobile phase is 0.05 mol / L potassium dihydrogen phosphate. The conditions are: flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL, detection duration 25 min, and detection wavelength 260 nm.

[0036] The pretreatment method of the enzyme reaction solution before liquid phase detection: Centrifuge at 9000 rpm for 3 min, aspirate the supernatant reaction solution with a syringe, pass it through a 0.22 μm water-based membrane and inject it into the liquid phase vial. Analyze the sample by high performance liquid chromatography. Using the peak emergence time of the NMN standard product as a reference, calculate the peak emergence area of NMN in the sample at the corresponding time, and calculate the content of NMN according to the standard curve.

[0037] Example 1 Expression and purification of pGEX-beanrk

[0038] The recombinant plasmid pGEX-beanrk synthesized by General Biology Co., Ltd. was transferred into Escherichia coli BL21(DE3) competent cells, and the BEANRK genetic engineering bacteria were screened. The transfer and induction expression of Escherichia coli are as follows: Prepare 300 mL of large-volume LB liquid medium, inoculate 1 mL of the activated culture solution, 300 μL of Amp, at 37 °C, 200 rpm, and culture for 2 h until the cell concentration OD 600When it reaches 0.4 - 0.6, IPTG with a final concentration of 25 mg / L is added for induction. Subsequently, low-temperature induction culture is carried out under the conditions of 20 °C, 150 rpm for 20 h. Subsequently, the wet bacterial cells are collected and resuspended in the prepared Escherichia coli lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol, pH 7). The bacterial suspension is sonicated with a sonication time of 5 s, an interval of 5 s, a power of 25%, and a total working time of 15 min. The lysate is centrifuged at high speed and low temperature, and the supernatant crude enzyme solution is collected. The Ni-NTA nickel column is used to purify the target protein with a His tag, and the imidazole eluate is collected and immediately ultrafiltered. After concentration, buffer is added for dilution, and this is repeated to remove imidazole from the pure enzyme solution, and the purity and concentration of the protein in it are detected.

[0039] Example 2 BEANRK Structure Modeling and Analysis

[0040] In order to obtain the structure of BEANRK from a new source, it is first necessary to perform homology modeling on NRK. Human HNRK is the only NRK crystal structure in the current PDB database. As Figure 1 shown, HNRK consists of a β-sheet containing five β-strands, with two α-helices on one side and a large helix on the other side. The five strands of the sheet are completely parallel, and the substrate binds below the Lid. In addition, the monomeric enzyme contains a Lid domain composed of two helices connected by a 12-amino acid loop. The sequence identity between BEANRK and HNRK is 19.22%. Compared with HNRK, the obtained BEANRK homology model has a high consistency with the template 2qt0 at the active site of substrate binding, but BEANRK has a longer Lid structure, and there is an additional long loop and an α-helix structure at the amino acid sequence from His78 to Leu127 ( Figure 1 ).

[0041] The rationality of the BEANRK modeling result is evaluated by drawing the Ramachandran plot of the model. As Figure 2 shown, the results show that 93.2% of the residues fall within the allowed region, and 5.8% of the amino acid residues are located in the additionally allowed region, indicating that the conformation of this model is reasonable and can be used as the tertiary structure model of BEANRK.

[0042] Example 3 Design of Single-Point Amino Acid Mutations

[0043] The sequences of BEANRK are aligned with KLUNRK and HNRK, and the amino acids that are unfavorable to the catalytic activity of BEANRK are mutated to improve the enzyme activity of the wild-type BEANRK from the new source, and at the same time, the functions and importance of the amino acids at the active site are investigated.

[0044] The docking program AutoDock Vina 1.2.3 was used to dock BEANRK with substrates NR and ATP, and the substrate docking site and Lid structure were selected. The amino acids within the range were selected as candidate mutation points, and then the amino acids were replaced by KLUNRK and HNRK as reference templates through sequence analysis. The results of the amino acid sequence comparison analysis of the three enzymes are shown in Figure 2. Figure 3 As shown, the highly conserved region is GxxxSGKT, which is a signature P-loop motif of nucleotide triphosphate-binding proteins, in which lysine residues play a major role in phosphate transfer, so the amino acids in this region do not change.

[0045] As shown in Table 1, 11 monomeric mutants were constructed based on the steric hindrance and hydrophobicity of amino acids.

[0046] Table 1 Selection and design of mutation point amino acids

[0047]

[0048] Example 4 Expression and purification of mutants

[0049] Primers were designed based on the single-point or combined mutation sites, as shown in Table 2. Whole-plasmid PCR was performed using the pGEX-beanrk plasmid as a template. Subsequently, 1 μL of DpnI and 5 μL of Cut Buffer were added to 50 μL of the amplified plasmid and incubated at 37°C for 45-60 minutes to digest the template. Finally, expression of the 11 mutants was purified according to the method described in Example 1, and the pure enzyme solution was collected.

[0050] Table 2 Primers related to mutation sites

[0051]

[0052]

[0053] Example 5 Study on enzyme activity and thermal stability of single-point mutants

[0054] The above 11 mutants were used to perform catalytic reactions, detect NMN production and calculate enzyme activity. The results are as follows: Figure 4As shown in the figure. Among them, the relative enzyme activity of the mutant V172K is better than that of the WT. The relative enzyme activities of the two mutants V172T and L174R are basically the same as that of the WT, and the relative enzyme activities of the remaining 8 mutants are worse than that of the WT. The temperature stability was investigated by treating the purified enzyme solution at 20°C, 30°C, 40°C, and 50°C for 30 min, and then detecting its enzyme activity. The relative enzyme activity was calculated based on the untreated enzyme. The results showed that the temperature stability of this enzyme is relatively poor. The enzyme activity of the WT decreased to less than half after treatment at temperatures above 30°C. Compared with the WT, the mutant F177Y has better temperature stability. It can still maintain 63% of the relative enzyme activity at 40°C, while the V172K mutant with the highest enzyme activity before can also maintain the enzyme activity above 80% at 20°C and 30°C, showing a certain improvement compared with the WT. In addition, L174R can also maintain a relatively high relative enzyme activity at 20°C and 30°C. The relative activities of other mutants did not change favorably at the four temperatures. The above results indicate that the activities of most mutants did not increase, only V172K increased by 1.2 times, and the temperature stability of V172K also improved to a certain extent.

[0055] Example 6 Study on the Enzyme Activity and Thermal Stability of Combinatorial Mutants

[0056] The enzyme activities of the combinatorial mutants were also investigated by the catalytic products, and the relative enzyme activities were still calculated based on the WT. As Figure 5 shown, the relative enzyme activities of both mutants M1 / G176S and M1 / N92D still decreased, which is consistent with the results of single-point mutations and also proves that the two amino acids G176 and N92 should be retained, which may play an important role in the catalytic synthesis of products. Subsequently, the mutation sites G170Y, L174R, and F177Y were respectively superimposed on the basis of M1 / N92D. M1 / N92D / G170Y still maintained a relatively low enzyme activity, while the relative enzyme activity of M1 / N92D / L174R was increased and basically equal to that of the wild-type enzyme. M1 / N92D / F177Y also restored a certain relative enzyme activity, indicating that the combination of N92D and L174R may have a positive effect on the catalysis of the enzyme. Subsequently, after treatment with a temperature gradient, all combinatorial mutants showed basically the same temperature stability as the WT.

[0057] Example 7 Application of Mutants in the Synthesis of NMN

[0058] The above-mentioned relatively favorable single-point mutations and combined mutations were further verified for NMN synthesis. WT, V172K, V172K / N92D, and V172K / N92D / L174R were re-expressed and purified, and catalysis was carried out in the same system. The NMN yield, enzyme activity, and conversion rate of NR to NMN were calculated. As shown in Table 3, the conversion rate of V172K reached 91% because of the increase in specific enzyme activity, which was higher than that of WT. This indicates that V172K achieved a certain improvement in catalytic efficiency, while that of V172K / N92D decreased, and V172K / N92D / L174R remained almost unchanged.

[0059] Table 3 Catalytic synthesis of NMN by WT and mutants

[0060]

[0061] Analysis of the docking results of the optimal mutants in Example 8

[0062] The structures of all mutants were predicted and modeled by Alphafold2. The obtained structural models were docked with the double substrate. The ones with low docking energy and reasonable positions were selected as the final docking results, and the PLIP website was used to predict the docking interaction forces. As Figure 6 shown, in the docking conformation of WT, the ATP has a greater folding curvature, while in M1, the ATP can unfold in the substrate channel. And compared with WT, the terminal phosphate group of ATP in M1 is closer to the substrate NR, and the shortening of the spatial distance is very beneficial to the transfer of phosphate groups between substrates. In M1, in addition to K172 providing an additional hydrogen bond, N92 and S93 also respectively form additional hydrogen bonds with NR and ATP, and S93 directly stabilizes the phosphate group of ATP. R164 forms a cation-π interaction force with ATP, and Y171 forms a π-π stacking interaction force with NR. The above are several specific interaction forces formed in M1, and they are evenly distributed around the substrate docking point. Therefore, the mutant and the substrate can establish a sufficient hydrogen bond network, and two new interaction forces are also added, which improves the stability of the substrate in the catalytic transition state, and the low-energy conformation is also beneficial to improving the catalytic efficiency, which is consistent with the results of the increase in enzyme activity and stability in the actual catalytic experiment.

Claims

1. A nicotinamide riboside kinase mutant, characterized in that, The mutant is obtained by mutating the 172nd amino acid residue in the amino acid sequence of nicotinamide riboside kinase from valine to lysine; The amino acid sequence of the nicotinamide riboside kinase is as shown in SEQ ID NO:

2.

2. Use of the mutant according to claim 1 in the synthesis of β-nicotinamide mononucleotide.

3. The application according to claim 2, wherein The mutant synthesizes β-nicotinamide mononucleotide in a single-enzyme catalysis system.

4. The application according to claim 2, wherein The mutant uses nicotinamide ribose as a substrate to synthesize β-nicotinamide mononucleotide.

5. The application according to claim 2, wherein The reaction system for the mutant to synthesize β-nicotinamide mononucleotide includes nicotinamide ribose, adenosine triphosphate, magnesium sulfate heptahydrate and mutant pure enzyme solution.

6. The application according to claim 5, characterized in that, The dosage of the substrate in the reaction system is 0.1 - 10 g / L.

7. The application according to claim 2, characterized in that, The mutant synthesizes β-nicotinamide mononucleotide at 20 - 30 °C.

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