CN116790550A - Nicotinamide ribokinase mutant and design method and application thereof - Google Patents

Abstract

The invention relates to a nicotinamide riboside kinase mutant, a design method and application thereof, wherein the mutant is obtained by mutating 172 th amino acid residue in an amino acid sequence of nicotinamide riboside kinase from valine to lysine; the amino acid sequence of the nicotinamide riboside kinase is shown in SEQ ID NO:2 shows that compared with the wild BEANRK, the BEANRK mutant constructed by the invention has the advantages that the enzyme activity is successfully improved by 1.2 times, the conversion rate of catalyzing NR to the product NMN in a single enzyme catalysis system without expensive phosphoribosyl pyrophosphate can reach 91 percent, the thermal stability of the mutant is also improved, an effective way is provided for improving the performance of nicotinamide riboside kinase, the production cost is reduced, and the application prospect is wide.

Description

Nicotinamide ribokinase mutant and design method and application thereof

Technical Field

The invention relates to the technical field of enzyme structure transformation and biosynthesis, in particular to a nicotinamide riboside kinase mutant and a design method and application thereof.

Background

Beta-nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is a natural bioactive nucleotide. As early as 1948, the production of NAD was studied in yeast cells by the Coelenberg of Asian ⁺ NMN was found to react with Adenosine Triphosphate (ATP) to form NAD ⁺ NMN is thus an in vivo synthesis of NAD ⁺ Is an important precursor substance of (a). NMN is a naturally occurring product of the reaction of a phosphate group with nucleosides containing ribose and nicotinamide, and therefore NMN is also a vitamin B3 derivative. The pharmacological activity of NMN is mainly based on NAD ⁺ Biosynthesis mediated, can improve the cause NAD ⁺ In many clinical studies, NMN administration improves mitochondrial function in different metabolic organs in the body, due to cardiovascular and cerebrovascular diseases caused by deficiency, senile dementia, type II diabetes and obesity induced by age and diet. There is increasing evidence that NMN has wide range of health care applications and pharmaceutical therapeutic value.

Currently, the main production method of NMN is the chemical synthesis method and the biological enzyme method. The chemical synthesis method mainly uses nicotinamide as a starting material, and is catalyzed and condensed with tetraacetyl ribose after being protected by trimethyl silane to obtain triacetyl nicotinamide riboside trifluoro methanesulfonate, the triacetyl nicotinamide riboside chloride is obtained by ion exchange, then the nicotinamide riboside chloride is obtained by alkaline hydrolysis crystallization, and finally NMN is obtained by phosphorylation. The method involves a plurality of organic reagents, is harmful to the environment, has fine and strict conditions and is not easy to control. In contrast, the biological enzyme method has high substrate specificity, high catalytic efficiency, mild reaction conditions, easy control and environmental friendliness. Therefore, the bio-enzyme method is widely used for the production of NMN.

The biological enzyme method production process is mainly divided into two types: the nicotinamide phosphoribosyl transferase is taken as a core and is divided into single enzyme catalysis and multi-enzyme catalysis, and the price is very high because phosphoribosyl pyrophosphate is involved in the single enzyme catalysis, and the substrate is more and the purification is difficult in the multi-enzyme catalysis, so that the nicotinamide phosphoribosyl transferase is not suitable for industrial production; the other type is an NMN production method which takes nicotinamide riboside (Nicotinamide riboside, NR) and ATP as substrate raw materials and reacts under the catalysis of nicotinamide riboside kinase (NR kinase, NRK) to obtain NMN, and has the advantages of high yield, high product purity and the like, thereby becoming a very potential NMN production method. However, the NRK species currently used for excavation are few, and the performance of enzymes is to be improved. CN115948365a discloses the application of nicotinamide riboside kinase of saccharomyces cerevisiae and mutants after encoding genes thereof in nicotinamide mononucleotide production, and the NMN conversion rate reaches more than 70%. Under the condition of single NRK, the ATP usage amount is large, the conversion rate is low, and the production cost is high. Therefore, in order to achieve industrial scale NMN production, efficient and stable catalytic enzymes are still highly sought to be mined and studied.

Disclosure of Invention

The invention provides a mutant obtained by modifying wild nicotinamide riboside kinase by utilizing semi-rational design, amino acid mutation and other technologies, the enzyme performance of the mutant is improved compared with that of the wild nicotinamide riboside kinase, and the mutant is applied to NMN production, so that the purposes of reducing the production cost and improving the NMN yield can be achieved, and the mutant is beneficial to industrial production.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a nicotinamide riboside kinase mutant obtained by mutating amino acid residue 172 in the amino acid sequence of nicotinamide riboside kinase from valine to lysine;

the amino acid sequence of the nicotinamide riboside kinase is shown in SEQ ID NO: 2. SEQ ID NO:1 is the nucleotide sequence thereof.

Another object of the present invention is to provide a method for designing the mutant, comprising:

according to the size of an active pocket of nicotinamide riboside kinase and the distribution of substrate channels, selecting nicotinamide riboside kinase with larger active pocket and long and narrow distribution of substrate channels as mutation basic enzyme;

carrying out homologous modeling on the mutation basic enzyme to obtain a tertiary structure model of the mutation basic enzyme;

molecular docking is carried out on a tertiary structure model of the mutant basic enzyme and substrates nicotinamide ribose and adenosine triphosphate, and a docking site and a Lid structure of the substrates and the enzyme are selectedDesigning single-point mutants and combined mutants by taking amino acids in the single-point mutant as alternative mutation points;

constructing a mutant to perform a catalytic reaction, and selecting an optimized mutant according to the enzyme activity and the thermal stability.

As a preferred embodiment, after homologous modeling, the modeling results are evaluated for rationality using a rawster's plot of the drawn model.

It is a further object of the present invention to provide the use of the mutant described above for the synthesis of β -nicotinamide mononucleotide.

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

As a preferred embodiment, the mutant synthesizes beta-nicotinamide mononucleotide by taking nicotinamide riboside as a substrate.

As a preferred embodiment, the reaction system for synthesizing the beta-nicotinamide mononucleotide by the mutant comprises nicotinamide riboside, adenosine triphosphate, magnesium sulfate heptahydrate and mutant pure enzyme solution.

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

As a preferred embodiment, the substrate is used in an amount of 0.1 to 10g/L in the reaction system.

As a preferred embodiment, the mutant synthesizes β -nicotinamide mononucleotide at 20-30 ℃.

The invention has the beneficial effects that:

(1) The invention is based on the structural analysis of the existing common nicotinamide riboside kinase database, and finds three NRKs which are reported and used by patents: HNRK, derived from homo sapiens (Human); KLUNRK, derived from kluyveromyces marxianus (Kluyveromyces marxianus); KPNRK, derived from pichia pastoris (Komagataella phaffii), all have a large active pocket near the P-loop motif and the substrate channels are also distributed in the active pocket. According to the structural characteristics, in the preliminary prediction structure of 57 heterologous NRKs, a novel NRK enzyme source with a large active pocket and long and narrow substrate channel distribution is selected, a beauveria bassiana (Beauveria bassiana) nicotinamide riboside kinase gene BEANRK is determined as a research object, the structure of the novel NRK enzyme source is modified, the optimal mutant M1 is an amino acid single-point mutation at the 172 th position, the mutation point of V172K changes the original valine into lysine, the enzyme activity of BEANRK is successfully improved by 1.2 times, and the enzyme stability is also improved to a certain extent at 20 ℃ and 30 ℃.

(2) The conversion rate of the substrate NR of the mutant can reach 91% in a single enzyme catalytic system without expensive phosphoribosyl pyrophosphate, and the industrial production cost is reduced.

(3) The invention provides a rational structural transformation thought, which can provide reference for subsequent re-excavation of similar enzymes. The specific method is mainly that a conserved sequence P ring motif in nicotinamide riboside kinase is discovered first to determine the catalytic activity of the enzyme. The enzyme structure is universal by selection based on the size of the active pocket and substrate channel of the P-ring motif.

(4) In the invention, a substrate, an enzyme docking site and a Lid structure are selectedAmino acids in the mutant are used as alternative mutation points, and then the sequence analysis is carried out, and KLUNRK and HNRK are used as certain reference templates to replace the amino acids, so that the optimal mutant M1 is obtained. Molecular docking shows ATP terminal phosphate groups in M1Closer to the substrate NR, the shortening of the spatial distance is advantageous for the transfer of phosphate groups between the substrates. Therefore, by sequence alignment and structural information analysis, the improvement of the performance of the enzyme by referring to the site amino acid of the excellent enzyme is an effective way for modifying the new enzyme.

Drawings

FIG. 1 is a comparison of a 2qt0 docking model and BEANRK tertiary structure; a: HNRK structural model 2qt0 in PDB database; b: BEANRK structural model.

FIG. 2 shows the analysis results of the Lagrank model.

FIG. 3 shows the amino acid alignment of BEANRK, KLUNRK and HNRK.

FIG. 4 is a comparison of relative enzyme activity and thermostability of single point mutants and WT.

FIG. 5 is a comparison of relative enzyme activity and thermostability of the combination mutant and WT, V172K.

FIG. 6 shows the force and force differential analysis between BEANRK (A.B) and M1 (C.D) and the substrate.

Detailed Description

In the examples, the enzyme reaction system used to evaluate the catalytic performance of the mutants was as follows:

the substrate was 5g/L NR,12.5mM magnesium sulfate heptahydrate, 28mM ATP, and the final protein concentration was 0.2g/L in pure enzyme solution, and reacted at 37℃for 5min at 900 rpm.

The reaction product was analyzed by liquid chromatography using a Dyan U-3000, and the mobile phase was 0.05mol/L potassium dihydrogen phosphate using a HC-C18 column under the following conditions: the flow rate is 0.8mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the detection time is 25min, and the detection wavelength is 260nm.

The enzyme reaction solution is used for the treatment method before liquid phase detection: centrifuging at 9000rpm for 3min, sucking the supernatant, pumping into a liquid phase vial through a 0.22 μm water system membrane, analyzing the sample by high performance liquid chromatography, calculating the peak area of NMN in the sample under corresponding time by taking the peak time of NMN standard as reference, and calculating the NMN content according to a standard curve.

Example 1 expression and purification of pGEX-beans

Recombinant plasmid synthesized by general biological limited publicpGEX-bean is transferred into competent E.coli BL21 (DE 3) and screened to obtain BEANRK genetic engineering bacteria. Coli transfer induction was expressed as follows: preparing 300mL of large-volume LB liquid medium, inoculating 1mL of activated culture bacterial liquid, 300 mu L of Amp,37 ℃ and 200rpm, and culturing for 2h until the bacterial concentration OD ₆₀₀ When reaching 0.4-0.6, IPTG with the final concentration of 25mg/L is added for induction. Then, low-temperature induction culture was performed under conditions of 20℃and 150rpm for 20 hours. Subsequently, the wet cells were collected and resuspended in a prepared E.coli disruption buffer (50mM Tris,300mM NaCl,10% glycerol, pH 7), the bacterial suspension was sonicated for 5 seconds, gap 5 seconds, power 25%, total working time 15 minutes. And (5) centrifuging the crushed liquid at a high speed and a low temperature, and collecting a supernatant crude enzyme liquid. Purifying target protein with His tag by Ni-NTA nickel column, collecting imidazole eluent, ultrafiltering immediately, concentrating, diluting with buffer solution, repeating the above steps, removing imidazole in pure enzyme solution, and detecting the purity and concentration of protein.

Example 2BEANRK Structure modeling and analysis

In order to obtain the structure of the new source BEANRK, it is first necessary to model the homology of NRK. Human HNRK is the only NRK crystal structure in the current PDB database, as shown in FIG. 1, HNRK consists of a beta sheet comprising five beta sheets, two alpha helices on one side and one large helix on the other side, five sheets being perfectly parallel, and substrates being bound in the lower part of Lid. Furthermore, the monomeric enzyme comprises a cap-like Lid domain consisting of two helices joined by a 12 amino acid loop. The sequence identity of BEANRK and HNRK was 19.22%, and the resulting BEANRK homology model had higher identity to template 2qt0 at the active site of substrate binding, but BEANRK had a longer Lid structure and a longer loop and an alpha helix structure at amino acid sequences His78 to Leu127 (FIG. 1).

The rationality of the BEANRK modeling results was evaluated by drawing a Lagrange plot of the model. As shown in FIG. 2, the results indicate that 93.2% of the residues fall within the allowed region and 5.8% of the amino acid residues are within the additional allowed region, which indicates that the model is conformationally reasonable and can be used as a model of BEANRK tertiary structure.

Example 3 design of Single Point amino acid mutations

And (3) comparing the sequences of BEANRK with KLUNRK and HNRK, and mutating amino acids which are unfavorable for the catalytic activity of BEANRK so as to promote the enzyme activity of the wild BEANRK of a new source and simultaneously examine the functions and the importance of the amino acids of the active site.

Docking BEANRK with substrates NR, ATP using docking program AutoDock Vina 1.2.3, selection of substrate docking site and Lid StructureAmino acids in the sequence are taken as alternative mutation points, and then KLUNRK and HNRK are taken as certain reference templates to replace the amino acids through sequence analysis. The amino acid sequence alignment of the three enzymes is shown in FIG. 3, where the highly conserved region is GxxxSGKT, a region that is a marker p-loop motif of nucleotide triphosphate binding proteins, where lysine residues play a major role in phosphotransfer and thus amino acids within this region are unchanged.

As shown in table 1, 11 monomer mutant sites were constructed based on steric hindrance and hydrophobicity of amino acids.

TABLE 1 selection and design of mutation point amino acids

EXAMPLE 4 expression and purification of mutants

Primers were designed based on single point mutation or combination mutation sites as shown in table 2. Full plasmid PCR was performed using pGEX-bean rk plasmid as template. Subsequently, 1. Mu.L of LDpnI, 5. Mu.L of LCut Buffer was added to the amplified 50. Mu.L of plasmid and reacted at 37℃for 45-60 minutes to digest the template. Finally, 11 mutants were purified by expression as described in example 1, and pure enzyme solutions were collected.

TABLE 2 mutation site related primers

EXAMPLE 5 Single Point mutant enzyme Activity and thermal stability Studies

The above 11 mutants were subjected to catalytic reaction, NMN production was measured and enzyme activity was calculated, and the results are shown in FIG. 4. Wherein the relative enzyme activity of the mutant V172K is superior to that of the WT, the relative enzyme activity of two mutants V172T, L174R is basically consistent with that of the WT, and the relative enzyme activities of the other 8 mutants are inferior to that of the WT. The temperature stability was examined by treating the purified enzyme solutions at 20℃and 30℃and 40℃and 50℃for 30 minutes, respectively, and then detecting the enzyme activities, and calculating the relative enzyme activities based on the untreated enzyme. The results showed that the enzyme temperature stability itself was poor, and the enzyme activity decreased to less than half after the WT was treated at 30℃or higher. Compared with the WT, the mutant F177Y has better temperature stability, can still keep 63% of relative enzyme activity at 40 ℃, and the V172K mutant with the highest enzyme activity can keep more than 80% of enzyme activity at 20 ℃ and 30 ℃ and has a certain improvement compared with the WT. In addition, L174R also maintains high relative enzyme activity at 20℃and 30 ℃. Other mutants did not have any favorable change in relative activity at 4 temperatures. The results show that the activity of the mutant is not improved mostly, only V172K is improved by 1.2 times, and the temperature stability of V172K is improved to a certain extent.

Example 6 combination mutant enzyme Activity and thermal stability Studies

The combinatorial mutants were also examined for their enzymatic activity via the catalytic product and relative enzymatic activity was still calculated on a WT basis. As shown in FIG. 5, the relative enzymatic activities of both mutants of M1/G176S and M1/N92D still appeared to be downhill, consistent with single point mutation results, demonstrating that both G176 and N92 amino acids should be retained, which may play an important role in catalyzing the synthesis of the product. Subsequently, G170Y, L174R, F Y mutation points were superimposed on the M1/N92D basis, respectively. The relative enzyme activity of M1/N92D/G170Y remains relatively low, while the relative enzyme activity of M1/N92D/L174R is increased, substantially the same as that of the wild-type enzyme. M1/N92D/F177Y also restored some relative enzymatic activity, indicating that the combination of N92D and L174R may have a positive effect on the catalysis of the enzyme. Subsequently, all combinatorial mutations exhibited substantially consistent temperature stability with WT upon treatment with a temperature gradient.

EXAMPLE 7 application of mutants to NMN Synthesis

Further NMN synthesis verification is carried out on the relatively favorable single-point mutation and combined mutation, and WT, V172K, V K/N92D and V172K/N92D/L174R are re-expressed and purified to catalyze the same system. The yield of NMN, enzyme activity and conversion rate of NR to NMN were calculated, and as shown in Table 3, V172K was 91% higher than that of WT because of the increased specific enzyme activity. This shows that V172K achieves a certain improvement in catalytic efficiency, while V172K/N92D is reduced, and V172K/N92D/L174R is almost unchanged.

TABLE 3 catalytic synthesis of NMN by WT and mutant

Example 8 optimal mutant docking outcome analysis

All mutant structures are subjected to predictive modeling through alpha fold2, the obtained structural model is butted with double substrates, the final butting result is selected, the butting energy is low, the position is reasonable, and the PLIP website is used for predicting the butting acting force, as shown in figure 6, in the butting conformation of the WT, the bending degree of ATP folding is larger, and the ATP in M1 can be spread in a substrate channel. And the ATP terminal phosphate group in M1 is closer to the substrate NR than WT, shortening of the spatial distance is very advantageous for transfer of phosphate groups between substrates. In M1, in addition to K172 additionally providing hydrogen bond formation, both N92 and S93 also additionally form hydrogen bonds with NR and ATP, respectively, and S93 directly stabilizes the phosphate group of ATP. R164 forms a cation-pi interaction with ATP and Y171 forms pi-pi stacking with NR. The above are all the several unique forces developed in M1 and are uniformly around the substrate docking point. Therefore, the mutant and the substrate can establish enough hydrogen bond network, two new acting forces are added, the stability of the substrate in the catalytic transition state is improved, and the low-energy conformation is also beneficial to improving the catalytic efficiency, which is consistent with the result of improving the enzyme activity and stability in the actual catalytic experiment.

Claims (10)

1. A nicotinamide riboside kinase mutant, which is characterized in that the mutant is obtained by mutating the 172 th 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 shown in SEQ ID NO: 2.

2. A method for designing a nicotinamide riboside kinase mutant, comprising:

according to the size of an active pocket of nicotinamide riboside kinase and the distribution of substrate channels, selecting nicotinamide riboside kinase with larger active pocket and long and narrow distribution of substrate channels as mutation basic enzyme;

carrying out homologous modeling on the mutation basic enzyme to obtain a tertiary structure model of the mutation basic enzyme;

molecular docking is carried out on a tertiary structure model of the mutant basic enzyme and substrates nicotinamide ribose and adenosine triphosphate, and amino acids in a substrate and enzyme docking site and a Lid structure 10A are selected as alternative mutation points to design single-point mutants and combined mutants;

constructing a mutant to perform a catalytic reaction, and selecting an optimized mutant according to the enzyme activity and the thermal stability.

3. The method according to claim 2, characterized in that after homologous modeling, the modeling results are plausible assessed using a rador graph of the drawn model.

4. Use of the mutant according to claim 1 for the synthesis of β -nicotinamide mononucleotide.

5. The use according to claim 4, wherein the mutant synthesizes β -nicotinamide mononucleotide in a single enzyme catalytic system.

6. The use according to claim 4, wherein the mutant synthesizes β -nicotinamide mononucleotide using nicotinamide riboside as a substrate.

7. The method according to claim 4, wherein the reaction system for synthesizing the beta-nicotinamide mononucleotide by the mutant comprises nicotinamide riboside, adenosine triphosphate, magnesium sulfate heptahydrate and mutant pure enzyme solution.

8. The use according to claim 7, wherein the amount of the mutant pure enzyme solution in the reaction system is 0.1-0.5 g/L of the final protein concentration.

9. The use according to claim 7, wherein the substrate is used in an amount of 0.1 to 10g/L in the reaction system.

10. The use according to claim 4, wherein the mutant synthesizes β -nicotinamide mononucleotide at 20-30 ℃.