Molecular modification method for improving specificity of aldehyde ketoreductase substrate

Abstract

The invention belongs to the technical field of biotechnology and enzyme engineering, and relates to a molecular modification method for improving aldehyde ketoreductase substrate specificity, which comprises the following steps: establishing a substrate library of aldehyde ketoreductase AKR13B3 and analyzing kinetic parameters; the method comprises the following steps: performing loop structure analysis and amino acid sequence comparison on a constructed composite structure of aldoketoreductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON to determine a mutation site, and screening mutants with increased substrate specificity; according to the invention, the geometrical shape of the inlet ring region of the AKR13B3 substrate binding bag is designed to change the substrate preference, so that the catalytic activity of the enzyme to a specific substrate is increased; in addition, a feasible scheme is provided for designing the aldoketoreductase with good substrate specificity, and huge hope is provided for developing efficient enzyme preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of aldehyde-keto reductase, and in particular relates to a molecular modification method for improving the substrate specificity of aldehyde-keto reductase. Background Art

[0002] Mycotoxin contamination poses a serious threat to ecosystems and human health. It is estimated that the livestock and aquaculture industries lose 1 billion tons of grain each year due to mycotoxin contamination of raw materials. It is estimated that the livestock and aquaculture industries lose 1 billion tons of grain each year due to mycotoxin contamination of raw materials. Among the various mycotoxins contaminated in crops worldwide, deoxynivalenol (DON) is the most frequently detected mycotoxin from Fusarium infection, which has a serious negative impact on both economy and safety. Therefore, for the sake of public health and feed / food safety, it is necessary to adopt effective strategies to reduce the toxicity of DON. Compared with physical and chemical detoxification methods, the most promising strategy for DON biodegradation is the enzyme cascade catalysis of dehydrogenases and aldo-keto reductases.

[0003] Recently, two DON-degrading enzymes (DADH and AKR13B3) were discovered from the Devosi strain A6-2-2. DADH can degrade DON into the less toxic -keto-DON, while AKR13B3 can degrade DON into the less toxic -keto-DON. AKR13B3 degrades 3-keto-DON into the relatively non-toxic 3-epi-DON. However, its substrate heterozygosity is worrying. AKR13B3 can not only degrade 3-keto-DON into 3-epi-DON, but also has higher catalytic activity towards certain aldehyde substrates. MTT analysis showed that the IC values ​​of 3-epi-DON, 3-keto-DON and DON were 2.34, 2.72, and 2.37, respectively. 50 The values ​​were 146, 1.24, and 0.409 μg / mL, respectively. In addition, each country has set a permissible limit for DON in food or feed. In the United States and China, the maximum content of DON in cereals is 1 mg / kg. In the European Union, the permissible limit of DON in cereal flour and corn is 0.75 mg / kg. In order to reduce the contamination of 3-keto-DON in food or feed, enzymatic hydrolysis is an environmentally friendly and efficient method; however, the limited catalytic efficiency and specificity of enzymes hinder their widespread industrial application. The identification of mutants with stronger substrate specificity and catalytic activity using protein engineering strategies will be of great significance to their important applications in the food and feed industries. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a molecular modification method for improving the substrate specificity of aldehyde-keto reductase.

[0005] In order to achieve the purpose of the present invention, the present invention will be implemented by adopting the following technical solutions.

[0006] This method was applied to analyze the composition and kinetic parameters of the substrate library of aldehyde-keto reductase AKR13B3, determine the structure of aldehyde-keto reductase AKR13B3, and perform molecular docking with the substrate 3-keto-DON(1a) to determine the complex structure of the enzyme and substrate; through loop structure analysis and amino acid sequence alignment, the mutation sites were determined, and combined mutants with increased substrate specificity were screened.

[0007] A molecular modification method for improving the substrate specificity of aldehyde-keto reductase comprises the following steps:

[0008] S1. Establishment of substrate library of aldehyde-keto reductase AKR13B3;

[0009] S2. Analyze the kinetic parameters of aldehyde-keto reductase AKR13B3 for different substrates by measuring the reaction rate at different substrate concentrations;

[0010] S3. Determine the protein structure of the complex of aldehyde-keto reductase AKR13B3 and coenzyme NADPH, and perform molecular docking with the substrate 3-keto-DON to construct the complex structure of aldehyde-keto reductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON;

[0011] S4, performing loop structure analysis on the composite structure to determine all loop structures around the substrate 3-keto-DON binding pocket, using an alanine scanning method to find potential mutation sites in the loopA, loopB, and loopC structures, selecting potential mutation sites that can reduce the steric hindrance effect and mutating them to valine and glycine; in addition, selecting non-conserved residues from loopD as mutation sites by amino acid sequence alignment;

[0012] S5. Combinatorial mutagenesis is performed on all mutation sites obtained in step S4 to obtain mutants that can improve the substrate specificity and catalytic efficiency of aldehyde-keto reductase.

[0013] As a preferred embodiment of the present invention, the substrate library includes 1a: 3-keto-DON, 1b: propionaldehyde, 1c: butyraldehyde, 1d: furfural, 1e: glutaraldehyde, and 1f: benzaldehyde.

[0014] As a preferred embodiment of the present invention, the kinetic parameters include K m , k cat , k cat / Km .

[0015] As a preferred embodiment of the present invention, the analysis of the kinetic parameters is completed by fitting the Michaelis-Menten equation using GraphPad Prism 8 software.

[0016] As a preferred embodiment of the present invention, the protein structure is obtained by prediction via the AlphaFold server.

[0017] As a preferred embodiment of the present invention, the three-dimensional structure of the substrate 3-keto-DON is obtained by Chem3D20.0 drawing software or ZINC database.

[0018] As a preferred embodiment of the present invention, the molecular docking is performed using AutoDock Vina 1.2.3 software.

[0019] As a preferred embodiment of the present invention, the mutation sites include F207, F211, I208, P212, P209, L213, W210, M251, L255, P253, I254, P255, T257, S258, K259, F25, M28, R29, L30, T31, D33, I35, W36, P38, P39, T95, S97, Q101, P103, P104, and L105.

[0020] As a preferred embodiment of the present invention, the amino acid sequence alignment is performed using the sequence of aldehyde-keto reductase AKR13B3 and the sequence of AKR13B2 of Devosia sp. D6-9 strain.

[0021] As a preferred embodiment of the present invention, the mutant is T31V / Q101A / P103G.

[0022] As a preferred embodiment of the present invention, the quantitative determination method of the specificity of the mutant of aldehyde-keto reductase AKR13B3 to substrate A1 / A2 is as follows:

[0023]

[0024] Beneficial Effects

[0025] The present invention provides a molecular modification method for improving the substrate specificity of aldehyde-keto reductase, and uses the method to analyze the composition and kinetic parameters of the aldehyde-keto reductase AKR13B3 substrate library, determine the structure of the aldehyde-keto reductase AKR13B3, and perform molecular docking with the substrate 3-keto-DON (1a) to determine the complex structure of the enzyme and the substrate; through loop structure analysis and amino acid sequence alignment, determine the mutation site, screen the combination mutant with increased substrate specificity, and finally determine an optimal combination mutant T31V / Q101A / P103G, in which the substrate specificity is increased by 31.94 times, and the catalytic activity is increased. The aldehyde-keto reductase mutant designed by the present invention can improve the specificity and catalytic efficiency of the substrate 3-keto-DON (1a), and has broad development prospects in the industrial application of developing enzyme detoxifiers. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The composition and kinetic parameter analysis diagram of the AKR13B3 substrate library of the present invention; wherein: (A) shows the process of AKR13B3 catalyzing 1a-f substrates to generate 2a-f products; (B) shows the k values ​​of each substrate respectively. cat and K m (C) The figure shows the k value of each substrate. cat / K m value;

[0027] Figure 2 The structural model of AKR13B3 and the selected key residues; wherein: (A) the figure shows the structure of AKR13B3 binding to NADPH and substrate 1a, wherein NADPH and substrate 1a are represented by stick structures respectively; (B) the figure shows loopA, loopB, loopC and loopD in blue, green, yellow and magenta respectively; (C) the figure shows the amino acid sequence alignment of AKR13B3 and AKR13B2; (D) the figure shows the screening of key mutation sites;

[0028] Figure 3 The construction of the mutant library and the evolution of substrate preference of the present invention; wherein: (A) shows the construction of the site-directed mutation library to reduce steric hindrance; (B) shows the evolution path of AKR13B3;

[0029] Figure 4 is the S of AKR13B3 and its variants described in the present invention A1 / A2 Values; where: (A) Figure shows the S of the variants obtained by alanine scanning 1a / 1f (B) shows the S values ​​of variants mutated to glycine and valine. 1a / 1f Value; (C) Figure shows the S of the combined variants 1a / 1f value. DETAILED DESCRIPTION

[0030] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0031] As an embodiment of the present invention, AKR13B3 substrate library composition and kinetic parameter analysis

[0032] (1) The substrate library used includes 1a: 3-keto-DON, 1b: propionaldehyde, 1c: butyraldehyde, 1d: furfural, 1e: glutaraldehyde, 1f: benzaldehyde, such as Figure 1 (A) shown.

[0033] (2) The kinetic parameters (K) of wild-type AKR13B3 for different substrates were evaluated by measuring the reaction rate at different substrate concentrations (from 1 to 500 μM). m , k cat , k cat / K m ). The Michaelis-Menten equation was fitted using GraphPad Prism 8 software, and the kinetic parameters were then evaluated, such as Figure 1 (B) shown.

[0034] As an example of the present invention, the structural model of AKR13B3 and the selected key residues.

[0035] (1) The AKR13B3 / NADPH complex was predicted by the AlphaFold server (https: / / alphafoldserver.com / ); the three-dimensional structure of the substrate 3-keto-DON was obtained by Chem3D 20.0 drawing software or ZINC database (http: / / zinc.docking.org). The amino acid sequence was aligned by Jalview (https: / / www.jalview.org / ). In the present invention, AutoDockVina 1.2.3 software was used for molecular docking work, such as Figure 2 (A) As shown; PyMol2.5.5 software was used to process and visualize the receptor protein before docking.

[0036] (2) The loop around the substrate binding pocket is determined by analyzing the enzyme / substrate / coenzyme complex structure. The three-dimensional structure shows that the four loops A, B, C, and D are located at the entrance of the active center. The key amino acid residues on the loop are then determined as key amino acid residues, such as Figure 2 (B) In addition, the sequence alignment of AKR13B3 and AKR13B2 (PDB: 8HNQ) of Devosia sp. D6-9 strain was completed, as shown in Figure 2(C) As shown. Predicting active sites that reduce steric hindrance effects is one of the important steps to change substrate specificity. Alanine scanning was used to search for potential mutation sites within ring A (residues 206-216), ring B (residues 251-259), and ring C (residues 25-39). In addition, considering the steric hindrance factor, glycine was excluded (glycine has the least steric hindrance and can only be mutated based on steric hindrance enhancement). Through structural analysis of the AKR13B3 / NADPH / 1a complex, 25 key residues in the A, B, and C rings were selected, as shown in Figure 2. Figure 2 (D) In ​​addition, amino acid sequence alignment showed that six non-conserved residues in loop D were selected as suitable mutation sites.

[0037] As an embodiment of the present invention, the construction of a mutant library and the evolution of substrate preference are described.

[0038] Using alanine scanning to find favorable mutation sites is a common and effective method. However, to change the catalytic substrate preference of AKR13B3, changes in the geometric configuration of the rings around the active center may require other strategies. Studies have shown that bulky substrate 1a with a multi-ring structure is more stable in the hydrophobic active pocket. There are two other principles for the selection of target mutagenesis modules: (a) avoid polar molecular interactions and exclude polar and charged amino acids; (b) reduce spatial hindrance and replace mutation sites with smaller amino acid letters. According to these principles, we first used alanine scanning to select potential favorable residues. Among them, 10 residues were selected to mutate to two other hydrophobic amino acids, valine (V) and glycine (G), such as Figure 3 (A) As shown. Combinatorial mutagenesis was performed on the excellent single-site variants identified by the above method, such as Figure 3 (B) to obtain mutants with stronger substrate specificity and catalytic activity.

[0039] As an embodiment of the present invention, AKR13B3 and its variants A1 / A2 value.

[0040] To accurately describe the preference of AKR13B3 and its variants for a specific substrate, a parameter (S A1 / A2 ), which is obtained by the ratio of the catalytic activity of substrate A1 to the catalytic activity of A2. A1 / A2 The higher the value, the more favored A1 is than A2. As expected, the loops near the substrate binding pocket provided some good mutagenesis sites. According to the alanine scanning strategy, ten single-site variants (F25A, T31A, P39A, S97A, Q101A, P103A, P104A, L105A, F211A, M251A) were obtained, all of which were superior to wild-type AKR13B3, such as Figure 4(A) As shown. Subsequently, through the substrate walking strategy, 9 unit variants (T31V, Q101V, Q101G, P103V, P103G, P104V, P104G, L105V, M251V) were found to outperform wild-type AKR13B3, as shown in Figure 4 Finally, according to S 1a / 1f Values, six variants (T31V, Q101A, Q101G, P103V, P103A, P103G) that were superior to the wild-type AKR13B3 were selected as candidate variants for combined mutations, such as Figure 3 (B) S of T31V (55.71%), Q101A (51.35%), Q101G (37.80%), P103V (44.41%), P103A (52.57%) and P103G (68.81%) 1a / 1f The values ​​were higher than those of wild-type AKR13B3 (7.22%). Figure 4 (A) Based on the initial modification of AKR13B3, the synergistic effects between the six superior unit variants were further explored, resulting in the generation of 11 double mutants. 1a / 1f The values ​​are significantly higher than those of the single variants, especially the excellent double variant T31V / P103G (167.34%), which is 2.4 times higher than that of variant P103G. Figure 4 (B) shows a significant synergistic enhancement effect. In the third round of mutagenesis, S 1a / 1f The highest value of T31V / P103G was selected to explore the possibility of improving the preference of substrate 1a. 1a / 1f The S values ​​of T31V / Q101A / P103G (230.58%) and T31V / Q101G / P103G (187.59%) were determined to be two superior triple variants, namely T31V / Q101A / P103G and T31V / Q101G / P103G. 1a / 1f The values ​​are 1.4 and 1.1 times of T31V / P103G (167.34%), respectively. Figure 4 (C) Therefore, this approach of residue design in loops near the active site may be a viable option to improve specific substrate preference.

[0041] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but the scope of the rights of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by a person skilled in the art without departing from the scope and essence of the present invention should be within the scope of the rights of the present invention.

Claims

1. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase, characterized in that: The steps include: S1. Establishment of substrate library of aldehyde-keto reductase AKR13B3; S2. Analyze the kinetic parameters of aldehyde-keto reductase AKR13B3 for different substrates by measuring the reaction rate at different substrate concentrations; S3. Determine the protein structure of the complex of aldehyde-keto reductase AKR13B3 and coenzyme NADPH, and perform molecular docking with the substrate 3-keto-DON to construct the complex structure of aldehyde-keto reductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON; S4, performing loop structure analysis on the composite structure to determine all loop structures around the substrate 3-keto-DON binding pocket, using an alanine scanning method to find potential mutation sites in the loopA, loopB, and loopC structures, selecting potential mutation sites that can reduce the steric hindrance effect and mutating them to valine and glycine; in addition, selecting non-conserved residues from loopD as mutation sites by amino acid sequence alignment; S5. Combinatorial mutagenesis is performed on all mutation sites obtained in step S4 to obtain mutants that can improve the substrate specificity and catalytic efficiency of aldehyde-keto reductase.

2. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The substrate library includes 1a: 3-keto-DON, 1b: propionaldehyde, 1c: butyraldehyde, 1d: furfural, 1e: glutaraldehyde, and 1f: benzaldehyde.

3. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The kinetic parameters include K m , k cat , k cat / K m .

4. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 3, characterized in that: The analysis of the kinetic parameters was completed by fitting the Michaelis-Menten equation using GraphPad Prism 8 software.

5. The molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The protein structure was predicted by the AlphaFold server.

6. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The three-dimensional structure of the substrate 3-keto-DON is obtained by Chem3D 20.0 drawing software or ZINC database.

7. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The molecular docking was performed using AutoDock Vina 1.2.3 software.

8. The molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The mutation sites include F207, F211, I208, P212, P209, L213, W210, M251, L255, P253, I254, P255, T257, S258, K259, F25, M28, R29, L30, T31, D33, I35, W36, P38, P39, T95, S97, Q101, P103, P104, and L105.

9. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The amino acid sequence alignment was performed using the sequence of aldehyde-keto reductase AKR13B3 and the sequence of AKR13B2 of Devosia sp. D6-9 strain.

10. The molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The mutant is T31V / Q101A / P103G.

11. A molecular modification method for improving the substrate specificity of aldehyde-keto reductase according to claim 1, characterized in that: The quantitative determination method of the specificity of the mutant of the aldehyde-keto reductase AKR13B3 to the substrate A1 / A2 is as follows:

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