CN113897342A - PAP high-specificity coupling enzyme YND mutant based on molecular docking, method and application - Google Patents
PAP high-specificity coupling enzyme YND mutant based on molecular docking, method and application Download PDFInfo
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- CN113897342A CN113897342A CN202111107706.4A CN202111107706A CN113897342A CN 113897342 A CN113897342 A CN 113897342A CN 202111107706 A CN202111107706 A CN 202111107706A CN 113897342 A CN113897342 A CN 113897342A
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Abstract
The invention discloses a coupling enzyme YND mutant with high specificity of 3',5' -adenosine diphosphate based on molecular docking, wherein the gene sequence of the YND mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the amino acid sequence of the YND mutant is SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8. The invention designs and transforms a coupling enzyme with strong PAP specificity by taking yeast 3',5' -diphosphatase with known crystal structure as a starting point. The modified enzyme is induced and expressed by an escherichia coli expression system, is stored in a storage solution, can maintain the activity for 3 months at the temperature of minus 80 ℃, is diluted and used by a corresponding buffer system, is convenient and can replace the reaction system, and solves the problem of limitation of a system buffer substance on the detection of ST activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PAP (adenosine 3',5' -diphosphate) high-specificity coupling enzyme YND mutant based on molecular docking, a method and application thereof.
Background
Sulfosulfonyltransferases (ST) are a class of enzymes responsible for catalyzing the transfer of sulfonyl (SO3) groups from the universal donor molecule adenosine 3 '-phosphate-5' -phosphate sulfate (PAPS) to carbohydrates, proteins, and a variety of other low molecular weight metabolites. Sulfonyltransferases are present in most organisms and all human tissues, mediating sulfation of different kinds of receptors to perform a variety of biological functions. It has now been discovered that in biological systems, sulfonyltransferases play a variety of important roles, including detoxification, signaling, and modulation of receptor binding. In the biological sulphation process, inorganic sulphate is first activated as a high-energy cofactor and then the sulphate group is transferred to the final receptor.
The methods for measuring the activity of ST include isotope labeling, spectrophotometry, fluorometry and mass spectrometry, which are not preferred methods for measuring the activity of ST because of their toxic effects on operators, their fluorescent absorption and fluorescence properties of not all substrates or products, and their expensive instruments. The most convenient assay at present is the coupled enzymatic reaction. In the course of research on sulfonyltransferase, 3'-phosphoadenosine-5' -phosphosulfate (PAPS) is generally used as a sulfonyl donor, PAPS loses sulfo groups under the catalysis of sulfonyltransferase to become 3',5' -adenosine diphosphate (3'-phosphoadenosine 5' -phosphoadenosine, PAP), the activity and enzyme activity of the sulfonyltransferase can be judged by quantifying the generated PAP, PAP is decomposed into 5 '-adenine nucleotide (Adenosine 5' -monophosphophosphate, AMP) and phosphate ions through enzyme catalysis, the PAP yield can be determined through the generated phosphate ions, and the activity of the sulfonyltransferase can be further determined, the whole process is as follows:
the enzyme activity kit for measuring the activity of the sulfonyl transferase on the market at present has the defects of high price, single buffer solution of a reaction system and the like, and becomes resistance for screening a large amount of sulfonyl transferase and corresponding substrates, in addition, the buffer system used by a reagent of the kit is Tris-Hcl, Tris can possibly become a competitive substrate of a sulfonyl receptor in certain reactions, and difficulty is brought to the specific screening of the sulfonyl transferase substrate.
At present, the following defects exist in the prior art:
1. the methods for measuring the activity of ST include isotope labeling, spectrophotometry, fluorometry and mass spectrometry, which are not preferred methods for measuring the activity of ST because of their toxic effects on operators, their fluorescent absorption and fluorescence properties of not all substrates or products, and their expensive instruments. The most convenient assay at present is the coupled enzymatic reaction.
2. The enzyme activity kit for measuring the activity of the sulfonyl transferase on the market at present is expensive.
3. The buffer solution of the reaction system is single, and the buffer solution becomes resistance to massive screening of the sulfonyl transferase and the corresponding substrate, and in addition, the buffer system used by the reagent of the kit is Tris-Hcl, and Tris may become a competitive substrate of a sulfonyl receptor in certain reactions, thereby bringing difficulty to specific screening of the sulfonyl transferase substrate.
4. The existing enzyme optimization method has certain blindness, namely mutation is carried out according to approximate binding sites, so that the screening experiment task is large in amount and is not necessarily accurate. The invention applies molecular docking technology to accurately obtain the amino acid of the binding site of the enzyme and the substrate, and carries out accurate mutation on the binding site, thereby greatly reducing the experimental task amount and ensuring the purpose and the directionality to be more clear.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a PAP (adenosine 3',5' -diphosphate) high-specificity coupling enzyme YND mutant based on molecular docking, a method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a coupling enzyme YND mutant with high specificity based on 3',5' -Adenosine Diphosphate (ADP) subjected to molecular docking is disclosed, wherein the gene sequence of the YND mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the amino acid sequence of the YND mutant is SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
Further, molecular docking software Vina1Used to design YND with high specificity for PAP substrate, the three-dimensional structure of YND was defined as PDB ID 1K9Y2The protein structure of (a); mutants of YND were constructed using a Discovery Studio Visualizer; both substrates PAP and PAPs were plotted using ChemDraw; in the butt-joint study, the lattice spacing was set to
The center (x, y, z) of the lattice point is (9.623, 16.925, -23.414), and the parameters of the lattice point are set as
The number of conformations obtained by docking was 20.
Further, the YND mutant is induced to express by an Escherichia coli expression system.
Further, the YND mutant can be stored in a storage solution and can maintain the activity for 3 months at-80 ℃, and the YND mutant can be diluted by a corresponding buffer system for use.
The application of the coupling enzyme YND mutant with high specificity based on the molecular docking 3',5' -diphosphonidine in the aspect of sulfuryl transferase determination is disclosed.
The preparation method of the coupling enzyme YND mutant with high specificity based on the molecular docking 3',5' -diphosphonidine comprises the following steps:
firstly, designing a YND gene obtained by NCBI, inserting XhoI and NcoI enzyme cutting sites at two ends of the gene, checking and utilizing degeneracy of codons to remove the XhoI and NcoI enzyme cutting sites existing on the gene, wherein the modified amino acid sequence is SEQ ID NO.5, and the gene sequence is SEQ ID NO. 1;
synthesizing a modified YND gene by a gene artificial synthesis method, connecting the obtained YND gene with a PET28a (+) linear vector subjected to restriction endonuclease digestion by XhoI and NcoI by using solution I ligase to construct an expression vector for expressing YND;
obtaining mutant DNA by using an overlapping PCR method, designing a site-directed mutation PCR primer according to a site needing mutation, which is obtained by butting a YND sequence and molecules, selecting a homologous sequence containing a mutated base 15-25bp by taking a mutant site as a center, selecting a base 17-30bp upstream and downstream as a primer, and designing primer sequences from SEQ ID NO.7 to SEQ ID NO. 16;
transforming the constructed mutant plasmid into E coli JM109 competent cells for amplification and carrying out sequencing verification on the cells;
transforming the mutation plasmid with successful sequencing into E coli BL21, selecting the transformed single colony to be cultured in a 5mLLB culture medium test tube overnight, inoculating the obtained bacterial liquid into a 1L triangular flask liquid culture medium for liquid culture, and culturing at 37 ℃ to OD600When the temperature is up to 0.6, IPTG is added and the mixture is placed in a shaking table at the temperature of 16 ℃ for induction expression; collecting thalli after 20 h;
taking lysate suspension thalli, carrying out ultrasonic disruption on the thalli in ice bath at 12000rpm for 30min, and collecting supernatant;
purifying target protein by nickel column affinity chromatography, ultrafiltering protein obtained by nickel column affinity chromatography with stock solution, and replacing buffer system to obtain YND mutant, and storing at-80 deg.C;
wherein the stock solution is 50Mm Hepes buffer solution, and the pH value is 7.5.
Further, the YND is yeast 3', 5-The enzyme adenosine diphosphate is added to the reaction mixture,www.pdb.org；PDB:1KA1。
the invention has the advantages and positive effects that:
1. the present invention relates to yeast 3',5' -diphosphonite (YND) having a known crystal structure (www.pdb.org(ii) a PDB:1KA1) as a starting point, a PAP specific coupling enzyme is designed and modified. The modified enzyme is induced and expressed by an escherichia coli expression system, is stored in a storage solution, can maintain the activity for 3 months at the temperature of minus 80 ℃, is diluted and used by a corresponding buffer system, is convenient and can replace the reaction system, and solves the problem of limitation of a system buffer substance on the detection of ST activity.
2. The method firstly calculates the possible forward mutation sites of the adenosine monophosphate YND with the known crystal structure through molecular docking design, and then carries out the modification of enzyme molecules based on the forward mutation sites, thereby greatly reducing the screening workload and greatly reducing the ST activity detection cost.
3. The method of the invention carries out mutation design based on molecular docking and is assisted with experimental verification to improve the substrate specificity of YND to PAP, and the purified protein can change the reaction system according to the experimental requirements. The invention takes Hepes as a buffer system, determines the activity of the Hepes, optimizes the conditions, and tests the application effect of the mutant enzyme in the activity determination system of the sulfonyltransferase by using the optimized reaction conditions.
Drawings
FIG. 1 is a diagram of the validation of overlapping PCR in the present invention; wherein, 1, PCR left arm; 714bp, 2 is the PCR right arm; 410bp, 3: overlapping PCR product; 1079bp, M is marker;
FIG. 2 is a restriction enzyme map according to the present invention; wherein, the plasmid is linearized by PET28a (+) 1: the weight ratio; 2, YND enzyme digestion fragment; 3:236D enzyme digestion fragment; 4:236Y enzyme cutting fragment; 5, 236W enzyme digestion fragment; m is marker;
FIG. 3 is a photograph of SDS-PAGE protein gel according to the present invention; wherein, YND is used for purifying protein; 2:236D purified protein; 3:236Y purified protein; 4:236W purified protein; m is protein marker;
FIG. 4 is a graph showing the use of the 236D mutein of the invention in sulfonyltransferases (visualization using malachite green-ammonium molybdate staining); wherein, the experimental group: 10uLPAPS +25uL ST +5uL 236D +10uL 1-naphthol, control 1: 10uL PAPS +25uL ST +5uL reaction solution +10uL 1-naphthol, control 2: 10uLPAPS +25uL reaction solution +5uL 236D +10uL 1-naphthol, control 3: 10uLPAPS +30uL reaction solution +10uL 1-naphthol.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A coupling enzyme YND mutant with high specificity based on 3',5' -Adenosine Diphosphate (ADP) subjected to molecular docking is disclosed, wherein the gene sequence of the YND mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the amino acid sequence of the YND mutant is SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
Preferably, the molecular docking software Vina1Used to design YND with high specificity for PAP substrate, the three-dimensional structure of YND was defined as PDB ID 1K9Y2The protein structure of (a); mutants of YND were constructed using a Discovery Studio Visualizer; both substrates PAP and PAPs were plotted using ChemDraw; in the butt-joint study, the lattice spacing was set to
The center (x, y, z) of the lattice point is (9.623, 16.925, -23.414), and the parameter of the lattice point is set to 25
The number of conformations obtained by docking was 20.
Preferably, the YND mutant is expressed by an E.coli expression system.
Preferably, the YND mutant is stored in a storage solution, can maintain the activity for 3 months at-80 ℃, and is diluted by a corresponding buffer system for use.
The application of the coupling enzyme YND mutant with high specificity based on the molecular docking 3',5' -diphosphonidine in the aspect of sulfuryl transferase determination is disclosed.
The preparation method of the coupling enzyme YND mutant with high specificity based on the molecular docking 3',5' -diphosphonidine comprises the following steps:
firstly, designing a YND gene obtained by NCBI, inserting XhoI and NcoI enzyme cutting sites at two ends of the gene, checking and utilizing degeneracy of codons to remove the XhoI and NcoI enzyme cutting sites existing on the gene, wherein the modified amino acid sequence is SEQ ID NO.5, and the gene sequence is SEQ ID NO. 1;
synthesizing a modified YND gene by a gene artificial synthesis method, connecting the obtained YND gene with a PET28a (+) linear vector subjected to restriction endonuclease digestion by XhoI and NcoI by using solution I ligase to construct an expression vector for expressing YND;
obtaining mutant DNA by using an overlapping PCR method, designing a site-directed mutation PCR primer according to a site needing mutation, which is obtained by butting a YND sequence and molecules, selecting a homologous sequence containing a mutated base 15-25bp by taking a mutant site as a center, selecting a base 17-30bp upstream and downstream as a primer, and designing primer sequences from SEQ ID NO.7 to SEQ ID NO. 16;
transforming the constructed mutant plasmid into E coli JM109 competent cells for amplification and carrying out sequencing verification on the cells;
transforming the mutation plasmid with successful sequencing into E coli BL21, selecting the transformed single colony to be cultured in a 5mLLB culture medium test tube overnight, inoculating the obtained bacterial liquid into a 1L triangular flask liquid culture medium for liquid culture, and culturing at 37 ℃ to OD600When the temperature is up to 0.6, IPTG is added and the mixture is placed in a shaking table at the temperature of 16 ℃ for induction expression; collecting thalli after 20 h;
taking lysate suspension thalli, carrying out ultrasonic disruption on the thalli in ice bath at 12000rpm for 30min, and collecting supernatant;
purifying target protein by nickel column affinity chromatography, ultrafiltering protein obtained by nickel column affinity chromatography with stock solution, and replacing buffer system to obtain YND mutant, and storing at-80 deg.C;
wherein the stock solution is 50Mm Hepes buffer solution, and the pH value is 7.5.
Preferably, the YND is yeast 3',5' -diphosphatase,www.pdb.org；PDB:1KA1。
specifically, the preparation is as follows:
molecular docking software Vina1Was used to design YND with higher specificity for PAP substrates. Three-dimensional Structure of YND Using PDB ID of 1K9Y2The protein structure of (1). Mutants of YND were constructed using the Discovery Studio Visualizer. In addition, the substrates PAP and PAPs were both plotted using ChemDraw. In the butt-joint study, the lattice spacing was set to
And the center (x, y, z) of the lattice point is (9.623, 16.925, -23.414). In addition, the grid point parameter is set as
The number of conformations obtained by docking was 20.
The saturation mutation of amino acids was determined by observing the three-dimensional structure of binding of PAP and YND as substrates. Here, the residues D263, E238, G236, H241, K267, Q265, S264, V237 and Y268 were subjected to full saturation mutagenesis. Subsequently, the substrates PAP and PAPS were subjected to docking studies with the wild-type and mutant-type target binding regions of YND, respectively. And (5) screening according to the final score of the molecular docking. Finally, YND mutants G236Y, G236D and G236W (mutant proteins such as 236Y, 236D and 236W) with higher specificity to PAP substrate are selected and screened out.
More specifically:
YND (Yeast 3',5' -biphosphate adenylase, YND) obtained from NCBI (Yeast 3',5' -biphosphate nucleotidase, YND)www.pdb.org(ii) a PDB:1KA1)) gene, inserting XhoI and NcoI enzyme cutting sites at two ends of the geneThe XhoI and NcoI cleavage sites present in the gene were deleted by checking and utilizing the degeneracy of codons, and the modified nucleotide sequence was shown as nucleotide sequence 1.
The modified YND gene is synthesized by a method of artificially synthesizing the gene, the obtained YND gene is connected with a PET28a (+) linear vector which is cut by XhoI and NcoI restriction endonuclease, and a solution I ligase is used for constructing an expression vector for expressing YND. The restriction enzyme digestion linear plasmid and the target gene are shown in a restriction enzyme digestion validation gel figure 2.
Obtaining mutant DNA by using an overlapping PCR method, designing a site-directed mutagenesis PCR primer according to a site needing mutagenesis, which is obtained by butting a YND sequence and molecules, selecting a homologous sequence containing mutated base about 15-25bp by taking a mutagenesis site as a center, selecting 17-30bp base upstream and downstream as a primer, and designing a primer as shown in a sequence table 1:
TABLE 1 primer sequences corresponding to single point mutations
And transforming the constructed mutant plasmid into E coli JM109 competent cells for amplification and sequencing verification.
Transforming E coli BL21 into the mutation plasmid with successful sequencing, selecting the transformed single colony and culturing the single colony overnight in a 5ml LB test tube, inoculating the obtained bacterial liquid into a 1L triangular flask for liquid culture, and culturing the bacterial liquid at 37 ℃ to OD600When the temperature is about 0.6 ℃, IPTG is added and the mixture is placed in a shaking table at the temperature of 16 ℃ for induction expression. After 20h, the cells were collected.
30ml of lysate is taken, thalli is suspended, and the thalli is ultrasonically crushed in ice bath. 12000rpm,30min, collect the supernatant.
Purifying target protein by nickel column affinity chromatography, ultrafiltering protein obtained by nickel column affinity chromatography with stock solution (50Mm Hepes buffer solution Ph7.5), replacing its buffer system, and storing at-80 deg.C for use. The target protein and mutant protein after ultrafiltration concentration are shown in SDS-PAGE protein electrophoresis gel of figure 3.
The correlation measurements were as follows:
1. the invention uses the combination energy as the judgment basis through the molecular docking and molecular simulation methods, the result shows that the YND specificity can be changed by mutating the amino acid at the 236 site, and the mutant gene is obtained by performing single-point mutation on the YND gene by the technical means of overlapping PCR. The results are shown in FIG. 1.
2. The invention optimizes the reaction conditions of the mutant protein with better effect, optimizes the reaction temperature, the pH value of the reaction system, the addition amount of additives such as glycerol and the addition of the enzyme activity protective agent BSA. The results are shown in tables 2 to 6.
TABLE 2 comparison of enzyme activity ratios of different muteins on two substrates PAPS and PAP
From the results, the enzyme activity ratio of the original protein YND to the two substrates is close to 1, namely, the specificity difference of the original protein YND to the two substrates under the same condition is not large, the activity of the original protein YND to the substrate PAP is larger, namely, the specificity of the original protein YND to the PAP is slightly stronger. The PAP specificity of the mutated protein is 236D,236W and 236Y in descending order, and all mutations are positive mutations, because the final aim is to screen the mutated protein with strong PAP specificity and then carry out condition optimization based on the optimal mutation 236D.
TABLE 3 Effect of reaction temperature on mutant protein 236D substrate specificity
TABLE 4 Effect of pH on mutein 236D substrate specificity
TABLE 5 Effect of Glycerol addition on mutein 236D substrate specificity
TABLE 6 Effect of BSA addition on mutein 236D substrate specificity
The present invention will facilitate the detection of the activity of the sulfonyltransferase and the screening of specific substrates.
236D is taken as an example to describe the point mutation and activity assay,
firstly, using artificially synthesized gene YND as template and primer F-YND,236Rd；236Fd,R-YNDObtaining the mutated left arm and right arm by PCR respectively, taking the obtained left arm and right arm as templates, F-YND,R-YNDThe complete mutant gene was obtained by performing overlap PCR for the primers. This gene was digested with restriction endonucleases XhoI and NcoI to remove the protected bases and cut cohesive ends, and simultaneously the PET28a (+) plasmid was linearized with the same restriction endonucleases. The linearized plasmid was ligated to the mutant gene by ligase solution I overnight at 16 ℃. And transforming the constructed mutant plasmid into E coli JM109 competent cells for amplification and sequencing verification.
Transforming E coli BL21 into the mutation plasmid with successful sequencing, selecting the transformed single colony and culturing the single colony overnight in a 5ml LB test tube, inoculating the obtained bacterial liquid into a 1L triangular flask for liquid culture, and culturing the bacterial liquid at 37 ℃ to OD600When the temperature is about 0.6 ℃, IPTG is added and the mixture is placed in a shaking table at the temperature of 16 ℃ for induction expression. After 20h, the cells were collected.
30ml of lysate is taken, thalli is suspended, and the thalli is ultrasonically crushed in ice bath. 12000rpm,30min, collect the supernatant.
Purifying the target protein by a nickel column affinity chromatography method, and replacing the purified protein with a storage solution by ultrafiltration for storage and later use.
The finally obtained protein was diluted and subjected to the subsequent reaction at a use concentration of 0.01 ug/uL.
1mMPAPS/1mMPAP 10uL,0.01ug/uL 236D protein 5uL, reaction solution ((50Mm Hepes buffer 50Mm KCl 2Mm MgCl)2pH7.5)) was added to the reaction solution, and the mixture was gently mixed with 35uL of the aqueous solution. The dilution of the substrate and protein used was carried out with the reaction solution in the process. The reaction conditions were 37 ℃ water bath, pH7.5, and reaction time 20 minutes. After the reaction is finished, 30uL of malachite green color developing agent A,100uL of membrane passing water and 30uL of malachite green color developing agent B are added into each hole, and the mixture is uniformly mixed and then stands for 20 minutes at room temperature for color development. The colorimetry was carried out at 620 nm. The absorbance values of different substrates after the colorimetry are used as the basis for screening, and the PAPS/PAP ratio is used as the basis for specificity judgment. A closer ratio to 1 indicates a closer activity to the two substrates, i.e.a smaller substrate specificity. A ratio closer to 0 indicates a lower PAPS activity on the substrate, while a higher PAP activity on the substrate, i.e.a higher specificity.
And (5) the results of primary screening are shown. All mutations were positive mutations, i.e. the specificity of the enzyme for the substrate PAP was improved. The invention further optimizes the reaction condition of 236D protein with best mutation effect.
Using Ph7.550Mm Hepes buffer 50Mm Kcl 2Mm MgCl2As a reaction solution, the reaction time was 20 minutes, and the reaction temperature was optimized, and as can be seen from Table 3, the results demonstrated that the temperature was lowered, the specificity of the enzyme to the substrate was improved, and the subsequent application of the enzyme in the enzyme activity assay of sulfonyltransferase was integrated, taking 20 ℃ as the optimum reaction temperature.
50Mm Kcl 2Mm MgCl in 50Mm Hepes buffer at 20 ℃ and different pH2The reaction solution was reacted for 20 minutes to optimize the pH of the reaction system. The results demonstrated that the enzyme had the highest specificity for the substrate at pH7.5, and therefore 50mM Kcl 2mM MgCl was used at pH7.550Mm Hepes buffer2And performing subsequent optimization as a reaction solution. As shown in table 4.
Different volumes of glycerol are added into the reaction liquid, the concentration of final glycerol content (volume) of 1/1000,1/100,2/100 and 5/100 is used as the reaction liquid, the reaction is carried out for 20 minutes at 20 ℃ and pH7.5 as the reaction condition for optimization, and the experimental result proves that the specificity of the enzyme to the substrate is the highest when the glycerol is not contained. As shown in table 5.
To pH7.550Mm Hepes 50Mm Kcl 2Mm MgCl2Bovine Serum Albumin (BSA) was added to make the final concentration 1mg/ml, and the reaction solution was subjected to a water bath at 20 ℃ for 20 minutes, and the results in Table 6 show that the addition of BSA improves the specificity of the enzyme for the substrate.
Is finally prepared at pH7.550Mm Hepes 50Mm KCl 2Mm MgCl2BSA 1mg/ml was used as a reaction system, a water bath at 20 ℃ was used as a reaction condition, and a reaction time of 20 minutes was used as an optimum reaction condition. Then the application effect of the mutant enzyme 236D in the activity determination and the substrate verification of the sulfonyl transferase is carried out on the basis of the above.
And the obtained mutant enzyme is tested by using the sulfonyl transferase which is already available in a laboratory and has a definite corresponding substrate, so that the application of the mutant enzyme 236D to the detection of the enzyme activity of the sulfonyl transferase is feasible.
The test system is as follows:
the experimental group replaced the above optimal reaction solution with the missing components of other control groups of 10uL 1mMPAPS +25uL 100ng/uL ST +5uL 0.01ug/uL 236D +10uL 2mM 1-naphthol, and the reaction solution was used as a dilution solution for the dilution of the components in the reaction. The reaction conditions were pH7.550Mm Hepes 50Mm KCl 2Mm MgCl2BSA 1mg/ml was used as a reaction system, and the reaction was carried out in a water bath at 20 ℃ for 20 minutes.
From the experimental results, it can be seen in FIG. 4 that the color difference between the experimental group and the control group 2 is significant, i.e., the substrate specificity of the mutant 236D 3',5' -diphosphatase is sufficient for the activity detection and substrate identification of the sulfonyltransferase. Controls 1,3 were used to determine the activity of 236D, as well as the purity of PAPS.
The present invention relates to gene sequences:
YND(CP036482)
CCATGGCATTGGAAAGAGAATTATTGGTTGCAACTCAAGCTGTACGAAAGGCGTCTTTATTGACTAAGAGAATTCAATCTGAAGTGATTTCTCACAAGGACTCCACTACTATTACCAAGAATGATAATTCTCCAGTAACCACAGGTGATTATGCTGCACAAACGATCATCATAAATGCTATCAAGAGCAATTTTCCTGATGATAAGGTAGTTGGTGAAGAATCCTCATCAGGATTGAGCGACGCATTCGTCTCAGGAATTTTAAACGAAATAAAAGCCAATGACGAAGTTTATAACAAGAATTATAAAAAGGATGATTTTCTGTTTACAAACGATCAGTTTCCGCTAAAATCTTTGGAGGACGTCAGGCAAATCATCGATTTCGGCAATTACGAAGGTGGTAGAAAAGGAAGATTTTGGTGTTTGGATCCTATTGACGGAACCAAGGGGTTTTTAAGAGGTGAACAGTTTGCAGTATGTCTGGCCTTAATTGTGGACGGTGTTGTTCAGCTTGGTTGTATTGGATGCCCCAACTTAGTTTTAAGTTCTTATGGGGCCCAAGATTTGAAAGGCCATGAGTCATTTGGTTATATCTTTCGTGCTGTTAGAGGTTTAGGTGCCTTCTATTCTCCATCTTCAGATGCAGAGTCATGGACCAAAATCCACGTTAGACACTTAAAAGACACTAAAGACATGATTACTTTAGAGGGAGTTGAAAAGGGACACTCCTCTCATGATGAACAAACTGCTATCAAAAACAAACTAAATATATCCAAATCTTTGCACTTGGATTCTCAAGCCAAGTACTGTTTGTTAGCATTGGGCTTAGCAGACGTATATTTACGTCTGCCTATCAAACTTTCTTACCAAGAAAAGATCTGGGACCATGCTGCAGGCAACGTTATTGTCCATGAAGCTGGAGGTATCCATACAGATGCAATGGAAGATGTTCCTCTAGACTTCGGTAACGGTAGAACGCTAGCTACGAAGGGAGTTATAGCGTCAAGTGGCCCACGCGAGTTACATGACTTGGTGGTGTCTACATCATGCGATGTCATTCAGTCAAGAAACGCCCTCGAG
236D
CCATGGCATTGGAAAGAGAATTATTGGTTGCAACTCAAGCTGTACGAAAGGCGTCTTTATTGACTAAGAGAATTCAATCTGAAGTGATTTCTCACAAGGACTCCACTACTATTACCAAGAATGATAATTCTCCAGTAACCACAGGTGATTATGCTGCACAAACGATCATCATAAATGCTATCAAGAGCAATTTTCCTGATGATAAGGTAGTTGGTGAAGAATCCTCATCAGGATTGAGCGACGCATTCGTCTCAGGAATTTTAAACGAAATAAAAGCCAATGACGAAGTTTATAACAAGAATTATAAAAAGGATGATTTTCTGTTTACAAACGATCAGTTTCCGCTAAAATCTTTGGAGGACGTCAGGCAAATCATCGATTTCGGCAATTACGAAGGTGGTAGAAAAGGAAGATTTTGGTGTTTGGATCCTATTGACGGAACCAAGGGGTTTTTAAGAGGTGAACAGTTTGCAGTATGTCTGGCCTTAATTGTGGACGGTGTTGTTCAGCTTGGTTGTATTGGATGCCCCAACTTAGTTTTAAGTTCTTATGGGGCCCAAGATTTGAAAGGCCATGAGTCATTTGGTTATATCTTTCGTGCTGTTAGAGGTTTAGGTGCCTTCTATTCTCCATCTTCAGATGCAGAGTCATGGACCAAAATCCACGTTAGACACTTAAAAGACACTAAAGACATGATTACTTTAGAGGACGTTGAAAAGGGACACTCCTCTCATGATGAACAAACTGCTATCAAAAACAAACTAAATATATCCAAATCTTTGCACTTGGATTCTCAAGCCAAGTACTGTTTGTTAGCATTGGGCTTAGCAGACGTATATTTACGTCTGCCTATCAAACTTTCTTACCAAGAAAAGATCTGGGACCATGCTGCAGGCAACGTTATTGTCCATGAAGCTGGAGGTATCCATACAGATGCAATGGAAGATGTTCCTCTAGACTTCGGTAACGGTAGAACGCTAGCTACGAAGGGAGTTATAGCGTCAAGTGGCCCACGCGAGTTACATGACTTGGTGGTGTCTACATCATGCGATGTCATTCAGTCAAGAAACGCCCTCGAG
236W
CCATGGCATTGGAAAGAGAATTATTGGTTGCAACTCAAGCTGTACGAAAGGCGTCTTTATTGACTAAGAGAATTCAATCTGAAGTGATTTCTCACAAGGACTCCACTACTATTACCAAGAATGATAATTCTCCAGTAACCACAGGTGATTATGCTGCACAAACGATCATCATAAATGCTATCAAGAGCAATTTTCCTGATGATAAGGTAGTTGGTGAAGAATCCTCATCAGGATTGAGCGACGCATTCGTCTCAGGAATTTTAAACGAAATAAAAGCCAATGACGAAGTTTATAACAAGAATTATAAAAAGGATGATTTTCTGTTTACAAACGATCAGTTTCCGCTAAAATCTTTGGAGGACGTCAGGCAAATCATCGATTTCGGCAATTACGAAGGTGGTAGAAAAGGAAGATTTTGGTGTTTGGATCCTATTGACGGAACCAAGGGGTTTTTAAGAGGTGAACAGTTTGCAGTATGTCTGGCCTTAATTGTGGACGGTGTTGTTCAGCTTGGTTGTATTGGATGCCCCAACTTAGTTTTAAGTTCTTATGGGGCCCAAGATTTGAAAGGCCATGAGTCATTTGGTTATATCTTTCGTGCTGTTAGAGGTTTAGGTGCCTTCTATTCTCCATCTTCAGATGCAGAGTCATGGACCAAAATCCACGTTAGACACTTAAAAGACACTAAAGACATGATTACTTTAGAGCCAGTTGAAAAGGGACACTCCTCTCATGATGAACAAACTGCTATCAAAAACAAACTAAATATATCCAAATCTTTGCACTTGGATTCTCAAGCCAAGTACTGTTTGTTAGCATTGGGCTTAGCAGACGTATATTTACGTCTGCCTATCAAACTTTCTTACCAAGAAAAGATCTGGGACCATGCTGCAGGCAACGTTATTGTCCATGAAGCTGGAGGTATCCATACAGATGCAATGGAAGATGTTCCTCTAGACTTCGGTAACGGTAGAACGCTAGCTACGAAGGGAGTTATAGCGTCAAGTGGCCCACGCGAGTTACATGACTTGGTGGTGTCTACATCATGCGATGTCATTCAGTCAAGAAACGCCCTCGAG
236Y
CCATGGCATTGGAAAGAGAATTATTGGTTGCAACTCAAGCTGTACGAAAGGCGTCTTTATTGACTAAGAGAATTCAATCTGAAGTGATTTCTCACAAGGACTCCACTACTATTACCAAGAATGATAATTCTCCAGTAACCACAGGTGATTATGCTGCACAAACGATCATCATAAATGCTATCAAGAGCAATTTTCCTGATGATAAGGTAGTTGGTGAAGAATCCTCATCAGGATTGAGCGACGCATTCGTCTCAGGAATTTTAAACGAAATAAAAGCCAATGACGAAGTTTATAACAAGAATTATAAAAAGGATGATTTTCTGTTTACAAACGATCAGTTTCCGCTAAAATCTTTGGAGGACGTCAGGCAAATCATCGATTTCGGCAATTACGAAGGTGGTAGAAAAGGAAGATTTTGGTGTTTGGATCCTATTGACGGAACCAAGGGGTTTTTAAGAGGTGAACAGTTTGCAGTATGTCTGGCCTTAATTGTGGACGGTGTTGTTCAGCTTGGTTGTATTGGATGCCCCAACTTAGTTTTAAGTTCTTATGGGGCCCAAGATTTGAAAGGCCATGAGTCATTTGGTTATATCTTTCGTGCTGTTAGAGGTTTAGGTGCCTTCTATTCTCCATCTTCAGATGCAGAGTCATGGACCAAAATCCACGTTAGACACTTAAAAGACACTAAAGACATGATTACTTTAGAGTACGTTGAAAAGGGACACTCCTCTCATGATGAACAAACTGCTATCAAAAACAAACTAAATATATCCAAATCTTTGCACTTGGATTCTCAAGCCAAGTACTGTTTGTTAGCATTGGGCTTAGCAGACGTATATTTACGTCTGCCTATCAAACTTTCTTACCAAGAAAAGATCTGGGACCATGCTGCAGGCAACGTTATTGTCCATGAAGCTGGAGGTATCCATACAGATGCAATGGAAGATGTTCCTCTAGACTTCGGTAACGGTAGAACGCTAGCTACGAAGGGAGTTATAGCGTCAAGTGGCCCACGCGAGTTACATGACTTGGTGGTGTCTACATCATGCGATGTCATTCAGTCAAGAAACGCCCTCGAG
the invention relates to amino acid sequences
YND(NP_014577.1)
MALERELLVATQAVRKASLLTKRIQSEVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEESSSGLSDAFVSGILNEIKANDEVYNKNYKKDDFLFTNDQFPLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLALIVDGVVQLGCIGCPNLVLSSYGAQDLKGHESFGYIFRAVRGLGAFYSPSSDAESWTKIHVRHLKDTKDMITLEGVEKGHSSHDEQTAIKNKLNISKSLHLDSQAKYCLLALGLADVYLRLPIKLSYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLATKGVIASSGPRELHDLVVSTSCDVIQSRNALE
236D
MALERELLVATQAVRKASLLTKRIQSEVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEESSSGLSDAFVSGILNEIKANDEVYNKNYKKDDFLFTNDQFPLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLALIVDGVVQLGCIGCPNLVLSSYGAQDLKGHESFGYIFRAVRGLGAFYSPSSDAESWTKIHVRHLKDTKDMITLEDVEKGHSSHDEQTAIKNKLNISKSLHLDSQAKYCLLALGLADVYLRLPIKLSYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLATKGVIASSGPRELHDLVVSTSCDVIQSRNALE
236W
MALERELLVATQAVRKASLLTKRIQSEVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEESSSGLSDAFVSGILNEIKANDEVYNKNYKKDDFLFTNDQFPLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLALIVDGVVQLGCIGCPNLVLSSYGAQDLKGHESFGYIFRAVRGLGAFYSPSSDAESWTKIHVRHLKDTKDMITLEWVEKGHSSHDEQTAIKNKLNISKSLHLDSQAKYCLLALGLADVYLRLPIKLSYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLATKGVIASSGPRELHDLVVSTSCDVIQSRNALE
236Y
MALERELLVATQAVRKASLLTKRIQSEVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEESSSGLSDAFVSGILNEIKANDEVYNKNYKKDDFLFTNDQFPLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLALIVDGVVQLGCIGCPNLVLSSYGAQDLKGHESFGYIFRAVRGLGAFYSPSSDAESWTKIHVRHLKDTKDMITLEYVEKGHSSHDEQTAIKNKLNISKSLHLDSQAKYCLLALGLADVYLRLPIKLSYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLATKGVIASSGPRELHDLVVSTSCDVIQSRNALE
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Sequence listing
<110> Tianjin science and technology university
<120> PAP high specificity coupling enzyme YND mutant based on molecular docking, method and application
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1079
<212> DNA/RNA
<213> modified YND (CP036482 Gene sequence Unknown)
<400> 1
ccatggcatt ggaaagagaa ttattggttg caactcaagc tgtacgaaag gcgtctttat 60
tgactaagag aattcaatct gaagtgattt ctcacaagga ctccactact attaccaaga 120
atgataattc tccagtaacc acaggtgatt atgctgcaca aacgatcatc ataaatgcta 180
tcaagagcaa ttttcctgat gataaggtag ttggtgaaga atcctcatca ggattgagcg 240
acgcattcgt ctcaggaatt ttaaacgaaa taaaagccaa tgacgaagtt tataacaaga 300
attataaaaa ggatgatttt ctgtttacaa acgatcagtt tccgctaaaa tctttggagg 360
acgtcaggca aatcatcgat ttcggcaatt acgaaggtgg tagaaaagga agattttggt 420
gtttggatcc tattgacgga accaaggggt ttttaagagg tgaacagttt gcagtatgtc 480
tggccttaat tgtggacggt gttgttcagc ttggttgtat tggatgcccc aacttagttt 540
taagttctta tggggcccaa gatttgaaag gccatgagtc atttggttat atctttcgtg 600
ctgttagagg tttaggtgcc ttctattctc catcttcaga tgcagagtca tggaccaaaa 660
tccacgttag acacttaaaa gacactaaag acatgattac tttagaggga gttgaaaagg 720
gacactcctc tcatgatgaa caaactgcta tcaaaaacaa actaaatata tccaaatctt 780
tgcacttgga ttctcaagcc aagtactgtt tgttagcatt gggcttagca gacgtatatt 840
tacgtctgcc tatcaaactt tcttaccaag aaaagatctg ggaccatgct gcaggcaacg 900
ttattgtcca tgaagctgga ggtatccata cagatgcaat ggaagatgtt cctctagact 960
tcggtaacgg tagaacgcta gctacgaagg gagttatagc gtcaagtggc ccacgcgagt 1020
tacatgactt ggtggtgtct acatcatgcg atgtcattca gtcaagaaac gccctcgag 1079
<210> 2
<211> 1079
<212> DNA/RNA
<213> Gene sequence of YND mutant 236D (Unknown)
<400> 2
ccatggcatt ggaaagagaa ttattggttg caactcaagc tgtacgaaag gcgtctttat 60
tgactaagag aattcaatct gaagtgattt ctcacaagga ctccactact attaccaaga 120
atgataattc tccagtaacc acaggtgatt atgctgcaca aacgatcatc ataaatgcta 180
tcaagagcaa ttttcctgat gataaggtag ttggtgaaga atcctcatca ggattgagcg 240
acgcattcgt ctcaggaatt ttaaacgaaa taaaagccaa tgacgaagtt tataacaaga 300
attataaaaa ggatgatttt ctgtttacaa acgatcagtt tccgctaaaa tctttggagg 360
acgtcaggca aatcatcgat ttcggcaatt acgaaggtgg tagaaaagga agattttggt 420
gtttggatcc tattgacgga accaaggggt ttttaagagg tgaacagttt gcagtatgtc 480
tggccttaat tgtggacggt gttgttcagc ttggttgtat tggatgcccc aacttagttt 540
taagttctta tggggcccaa gatttgaaag gccatgagtc atttggttat atctttcgtg 600
ctgttagagg tttaggtgcc ttctattctc catcttcaga tgcagagtca tggaccaaaa 660
tccacgttag acacttaaaa gacactaaag acatgattac tttagaggac gttgaaaagg 720
gacactcctc tcatgatgaa caaactgcta tcaaaaacaa actaaatata tccaaatctt 780
tgcacttgga ttctcaagcc aagtactgtt tgttagcatt gggcttagca gacgtatatt 840
tacgtctgcc tatcaaactt tcttaccaag aaaagatctg ggaccatgct gcaggcaacg 900
ttattgtcca tgaagctgga ggtatccata cagatgcaat ggaagatgtt cctctagact 960
tcggtaacgg tagaacgcta gctacgaagg gagttatagc gtcaagtggc ccacgcgagt 1020
tacatgactt ggtggtgtct acatcatgcg atgtcattca gtcaagaaac gccctcgag 1079
<210> 3
<211> 1079
<212> DNA/RNA
<213> Gene sequence of YND mutant 236W (Unknown)
<400> 3
ccatggcatt ggaaagagaa ttattggttg caactcaagc tgtacgaaag gcgtctttat 60
tgactaagag aattcaatct gaagtgattt ctcacaagga ctccactact attaccaaga 120
atgataattc tccagtaacc acaggtgatt atgctgcaca aacgatcatc ataaatgcta 180
tcaagagcaa ttttcctgat gataaggtag ttggtgaaga atcctcatca ggattgagcg 240
acgcattcgt ctcaggaatt ttaaacgaaa taaaagccaa tgacgaagtt tataacaaga 300
attataaaaa ggatgatttt ctgtttacaa acgatcagtt tccgctaaaa tctttggagg 360
acgtcaggca aatcatcgat ttcggcaatt acgaaggtgg tagaaaagga agattttggt 420
gtttggatcc tattgacgga accaaggggt ttttaagagg tgaacagttt gcagtatgtc 480
tggccttaat tgtggacggt gttgttcagc ttggttgtat tggatgcccc aacttagttt 540
taagttctta tggggcccaa gatttgaaag gccatgagtc atttggttat atctttcgtg 600
ctgttagagg tttaggtgcc ttctattctc catcttcaga tgcagagtca tggaccaaaa 660
tccacgttag acacttaaaa gacactaaag acatgattac tttagagcca gttgaaaagg 720
gacactcctc tcatgatgaa caaactgcta tcaaaaacaa actaaatata tccaaatctt 780
tgcacttgga ttctcaagcc aagtactgtt tgttagcatt gggcttagca gacgtatatt 840
tacgtctgcc tatcaaactt tcttaccaag aaaagatctg ggaccatgct gcaggcaacg 900
ttattgtcca tgaagctgga ggtatccata cagatgcaat ggaagatgtt cctctagact 960
tcggtaacgg tagaacgcta gctacgaagg gagttatagc gtcaagtggc ccacgcgagt 1020
tacatgactt ggtggtgtct acatcatgcg atgtcattca gtcaagaaac gccctcgag 1079
<210> 4
<211> 1079
<212> DNA/RNA
<213> Gene sequence of YND mutant 236Y (Unknown)
<400> 4
ccatggcatt ggaaagagaa ttattggttg caactcaagc tgtacgaaag gcgtctttat 60
tgactaagag aattcaatct gaagtgattt ctcacaagga ctccactact attaccaaga 120
atgataattc tccagtaacc acaggtgatt atgctgcaca aacgatcatc ataaatgcta 180
tcaagagcaa ttttcctgat gataaggtag ttggtgaaga atcctcatca ggattgagcg 240
acgcattcgt ctcaggaatt ttaaacgaaa taaaagccaa tgacgaagtt tataacaaga 300
attataaaaa ggatgatttt ctgtttacaa acgatcagtt tccgctaaaa tctttggagg 360
acgtcaggca aatcatcgat ttcggcaatt acgaaggtgg tagaaaagga agattttggt 420
gtttggatcc tattgacgga accaaggggt ttttaagagg tgaacagttt gcagtatgtc 480
tggccttaat tgtggacggt gttgttcagc ttggttgtat tggatgcccc aacttagttt 540
taagttctta tggggcccaa gatttgaaag gccatgagtc atttggttat atctttcgtg 600
ctgttagagg tttaggtgcc ttctattctc catcttcaga tgcagagtca tggaccaaaa 660
tccacgttag acacttaaaa gacactaaag acatgattac tttagagtac gttgaaaagg 720
gacactcctc tcatgatgaa caaactgcta tcaaaaacaa actaaatata tccaaatctt 780
tgcacttgga ttctcaagcc aagtactgtt tgttagcatt gggcttagca gacgtatatt 840
tacgtctgcc tatcaaactt tcttaccaag aaaagatctg ggaccatgct gcaggcaacg 900
ttattgtcca tgaagctgga ggtatccata cagatgcaat ggaagatgtt cctctagact 960
tcggtaacgg tagaacgcta gctacgaagg gagttatagc gtcaagtggc ccacgcgagt 1020
tacatgactt ggtggtgtct acatcatgcg atgtcattca gtcaagaaac gccctcgag 1079
<210> 5
<211> 359
<212> PRT
<213> YND（NP_014577.1）(Unknown)
<400> 5
Met Ala Leu Glu Arg Glu Leu Leu Val Ala Thr Gln Ala Val Arg Lys
1 5 10 15
Ala Ser Leu Leu Thr Lys Arg Ile Gln Ser Glu Val Ile Ser His Lys
20 25 30
Asp Ser Thr Thr Ile Thr Lys Asn Asp Asn Ser Pro Val Thr Thr Gly
35 40 45
Asp Tyr Ala Ala Gln Thr Ile Ile Ile Asn Ala Ile Lys Ser Asn Phe
50 55 60
Pro Asp Asp Lys Val Val Gly Glu Glu Ser Ser Ser Gly Leu Ser Asp
65 70 75 80
Ala Phe Val Ser Gly Ile Leu Asn Glu Ile Lys Ala Asn Asp Glu Val
85 90 95
Tyr Asn Lys Asn Tyr Lys Lys Asp Asp Phe Leu Phe Thr Asn Asp Gln
100 105 110
Phe Pro Leu Lys Ser Leu Glu Asp Val Arg Gln Ile Ile Asp Phe Gly
115 120 125
Asn Tyr Glu Gly Gly Arg Lys Gly Arg Phe Trp Cys Leu Asp Pro Ile
130 135 140
Asp Gly Thr Lys Gly Phe Leu Arg Gly Glu Gln Phe Ala Val Cys Leu
145 150 155 160
Ala Leu Ile Val Asp Gly Val Val Gln Leu Gly Cys Ile Gly Cys Pro
165 170 175
Asn Leu Val Leu Ser Ser Tyr Gly Ala Gln Asp Leu Lys Gly His Glu
180 185 190
Ser Phe Gly Tyr Ile Phe Arg Ala Val Arg Gly Leu Gly Ala Phe Tyr
195 200 205
Ser Pro Ser Ser Asp Ala Glu Ser Trp Thr Lys Ile His Val Arg His
210 215 220
Leu Lys Asp Thr Lys Asp Met Ile Thr Leu Glu Gly Val Glu Lys Gly
225 230 235 240
His Ser Ser His Asp Glu Gln Thr Ala Ile Lys Asn Lys Leu Asn Ile
245 250 255
Ser Lys Ser Leu His Leu Asp Ser Gln Ala Lys Tyr Cys Leu Leu Ala
260 265 270
Leu Gly Leu Ala Asp Val Tyr Leu Arg Leu Pro Ile Lys Leu Ser Tyr
275 280 285
Gln Glu Lys Ile Trp Asp His Ala Ala Gly Asn Val Ile Val His Glu
290 295 300
Ala Gly Gly Ile His Thr Asp Ala Met Glu Asp Val Pro Leu Asp Phe
305 310 315 320
Gly Asn Gly Arg Thr Leu Ala Thr Lys Gly Val Ile Ala Ser Ser Gly
325 330 335
Pro Arg Glu Leu His Asp Leu Val Val Ser Thr Ser Cys Asp Val Ile
340 345 350
Gln Ser Arg Asn Ala Leu Glu
355
<210> 6
<211> 359
<212> PRT
<213> amino acid sequence of YND mutant 236D (Unknown)
<400> 6
Met Ala Leu Glu Arg Glu Leu Leu Val Ala Thr Gln Ala Val Arg Lys
1 5 10 15
Ala Ser Leu Leu Thr Lys Arg Ile Gln Ser Glu Val Ile Ser His Lys
20 25 30
Asp Ser Thr Thr Ile Thr Lys Asn Asp Asn Ser Pro Val Thr Thr Gly
35 40 45
Asp Tyr Ala Ala Gln Thr Ile Ile Ile Asn Ala Ile Lys Ser Asn Phe
50 55 60
Pro Asp Asp Lys Val Val Gly Glu Glu Ser Ser Ser Gly Leu Ser Asp
65 70 75 80
Ala Phe Val Ser Gly Ile Leu Asn Glu Ile Lys Ala Asn Asp Glu Val
85 90 95
Tyr Asn Lys Asn Tyr Lys Lys Asp Asp Phe Leu Phe Thr Asn Asp Gln
100 105 110
Phe Pro Leu Lys Ser Leu Glu Asp Val Arg Gln Ile Ile Asp Phe Gly
115 120 125
Asn Tyr Glu Gly Gly Arg Lys Gly Arg Phe Trp Cys Leu Asp Pro Ile
130 135 140
Asp Gly Thr Lys Gly Phe Leu Arg Gly Glu Gln Phe Ala Val Cys Leu
145 150 155 160
Ala Leu Ile Val Asp Gly Val Val Gln Leu Gly Cys Ile Gly Cys Pro
165 170 175
Asn Leu Val Leu Ser Ser Tyr Gly Ala Gln Asp Leu Lys Gly His Glu
180 185 190
Ser Phe Gly Tyr Ile Phe Arg Ala Val Arg Gly Leu Gly Ala Phe Tyr
195 200 205
Ser Pro Ser Ser Asp Ala Glu Ser Trp Thr Lys Ile His Val Arg His
210 215 220
Leu Lys Asp Thr Lys Asp Met Ile Thr Leu Glu Asp Val Glu Lys Gly
225 230 235 240
His Ser Ser His Asp Glu Gln Thr Ala Ile Lys Asn Lys Leu Asn Ile
245 250 255
Ser Lys Ser Leu His Leu Asp Ser Gln Ala Lys Tyr Cys Leu Leu Ala
260 265 270
Leu Gly Leu Ala Asp Val Tyr Leu Arg Leu Pro Ile Lys Leu Ser Tyr
275 280 285
Gln Glu Lys Ile Trp Asp His Ala Ala Gly Asn Val Ile Val His Glu
290 295 300
Ala Gly Gly Ile His Thr Asp Ala Met Glu Asp Val Pro Leu Asp Phe
305 310 315 320
Gly Asn Gly Arg Thr Leu Ala Thr Lys Gly Val Ile Ala Ser Ser Gly
325 330 335
Pro Arg Glu Leu His Asp Leu Val Val Ser Thr Ser Cys Asp Val Ile
340 345 350
Gln Ser Arg Asn Ala Leu Glu
355
<210> 7
<211> 359
<212> PRT
<213> amino acid sequence of YND mutant 236W (Unknown)
<400> 7
Met Ala Leu Glu Arg Glu Leu Leu Val Ala Thr Gln Ala Val Arg Lys
1 5 10 15
Ala Ser Leu Leu Thr Lys Arg Ile Gln Ser Glu Val Ile Ser His Lys
20 25 30
Asp Ser Thr Thr Ile Thr Lys Asn Asp Asn Ser Pro Val Thr Thr Gly
35 40 45
Asp Tyr Ala Ala Gln Thr Ile Ile Ile Asn Ala Ile Lys Ser Asn Phe
50 55 60
Pro Asp Asp Lys Val Val Gly Glu Glu Ser Ser Ser Gly Leu Ser Asp
65 70 75 80
Ala Phe Val Ser Gly Ile Leu Asn Glu Ile Lys Ala Asn Asp Glu Val
85 90 95
Tyr Asn Lys Asn Tyr Lys Lys Asp Asp Phe Leu Phe Thr Asn Asp Gln
100 105 110
Phe Pro Leu Lys Ser Leu Glu Asp Val Arg Gln Ile Ile Asp Phe Gly
115 120 125
Asn Tyr Glu Gly Gly Arg Lys Gly Arg Phe Trp Cys Leu Asp Pro Ile
130 135 140
Asp Gly Thr Lys Gly Phe Leu Arg Gly Glu Gln Phe Ala Val Cys Leu
145 150 155 160
Ala Leu Ile Val Asp Gly Val Val Gln Leu Gly Cys Ile Gly Cys Pro
165 170 175
Asn Leu Val Leu Ser Ser Tyr Gly Ala Gln Asp Leu Lys Gly His Glu
180 185 190
Ser Phe Gly Tyr Ile Phe Arg Ala Val Arg Gly Leu Gly Ala Phe Tyr
195 200 205
Ser Pro Ser Ser Asp Ala Glu Ser Trp Thr Lys Ile His Val Arg His
210 215 220
Leu Lys Asp Thr Lys Asp Met Ile Thr Leu Glu Trp Val Glu Lys Gly
225 230 235 240
His Ser Ser His Asp Glu Gln Thr Ala Ile Lys Asn Lys Leu Asn Ile
245 250 255
Ser Lys Ser Leu His Leu Asp Ser Gln Ala Lys Tyr Cys Leu Leu Ala
260 265 270
Leu Gly Leu Ala Asp Val Tyr Leu Arg Leu Pro Ile Lys Leu Ser Tyr
275 280 285
Gln Glu Lys Ile Trp Asp His Ala Ala Gly Asn Val Ile Val His Glu
290 295 300
Ala Gly Gly Ile His Thr Asp Ala Met Glu Asp Val Pro Leu Asp Phe
305 310 315 320
Gly Asn Gly Arg Thr Leu Ala Thr Lys Gly Val Ile Ala Ser Ser Gly
325 330 335
Pro Arg Glu Leu His Asp Leu Val Val Ser Thr Ser Cys Asp Val Ile
340 345 350
Gln Ser Arg Asn Ala Leu Glu
355
<210> 8
<211> 359
<212> PRT
<213> amino acid sequence of YND mutant 236Y (Unknown)
<400> 8
Met Ala Leu Glu Arg Glu Leu Leu Val Ala Thr Gln Ala Val Arg Lys
1 5 10 15
Ala Ser Leu Leu Thr Lys Arg Ile Gln Ser Glu Val Ile Ser His Lys
20 25 30
Asp Ser Thr Thr Ile Thr Lys Asn Asp Asn Ser Pro Val Thr Thr Gly
35 40 45
Asp Tyr Ala Ala Gln Thr Ile Ile Ile Asn Ala Ile Lys Ser Asn Phe
50 55 60
Pro Asp Asp Lys Val Val Gly Glu Glu Ser Ser Ser Gly Leu Ser Asp
65 70 75 80
Ala Phe Val Ser Gly Ile Leu Asn Glu Ile Lys Ala Asn Asp Glu Val
85 90 95
Tyr Asn Lys Asn Tyr Lys Lys Asp Asp Phe Leu Phe Thr Asn Asp Gln
100 105 110
Phe Pro Leu Lys Ser Leu Glu Asp Val Arg Gln Ile Ile Asp Phe Gly
115 120 125
Asn Tyr Glu Gly Gly Arg Lys Gly Arg Phe Trp Cys Leu Asp Pro Ile
130 135 140
Asp Gly Thr Lys Gly Phe Leu Arg Gly Glu Gln Phe Ala Val Cys Leu
145 150 155 160
Ala Leu Ile Val Asp Gly Val Val Gln Leu Gly Cys Ile Gly Cys Pro
165 170 175
Asn Leu Val Leu Ser Ser Tyr Gly Ala Gln Asp Leu Lys Gly His Glu
180 185 190
Ser Phe Gly Tyr Ile Phe Arg Ala Val Arg Gly Leu Gly Ala Phe Tyr
195 200 205
Ser Pro Ser Ser Asp Ala Glu Ser Trp Thr Lys Ile His Val Arg His
210 215 220
Leu Lys Asp Thr Lys Asp Met Ile Thr Leu Glu Tyr Val Glu Lys Gly
225 230 235 240
His Ser Ser His Asp Glu Gln Thr Ala Ile Lys Asn Lys Leu Asn Ile
245 250 255
Ser Lys Ser Leu His Leu Asp Ser Gln Ala Lys Tyr Cys Leu Leu Ala
260 265 270
Leu Gly Leu Ala Asp Val Tyr Leu Arg Leu Pro Ile Lys Leu Ser Tyr
275 280 285
Gln Glu Lys Ile Trp Asp His Ala Ala Gly Asn Val Ile Val His Glu
290 295 300
Ala Gly Gly Ile His Thr Asp Ala Met Glu Asp Val Pro Leu Asp Phe
305 310 315 320
Gly Asn Gly Arg Thr Leu Ala Thr Lys Gly Val Ile Ala Ser Ser Gly
325 330 335
Pro Arg Glu Leu His Asp Leu Val Val Ser Thr Ser Cys Asp Val Ile
340 345 350
Gln Ser Arg Asn Ala Leu Glu
355
<210> 9
<211> 24
<212> DNA/RNA
<213> R-YND(Unknown)
<400> 9
ccgctcgagg gcgtttcttg actg 24
<210> 10
<211> 29
<212> DNA/RNA
<213> F-YND(Unknown)
<400> 10
catgccatgg cattggaaag agaattatt 29
<210> 11
<211> 52
<212> DNA/RNA
<213> 236Rd(Unknown)
<400> 11
caacgtcctc taaagtaatc atgtctttag tgtcttttaa gtgtctaacg tg 52
<210> 12
<211> 45
<212> DNA/RNA
<213> 236Fd(Unknown)
<400> 12
gattacttta gaggacgttg aaaagggaca ctcctctcat gatga 45
<210> 13
<211> 52
<212> DNA/RNA
<213> 236Rw(Unknown)
<400> 13
caacccactc taaagtaatc atgtctttag tgtcttttaa gtgtctaacg tg 52
<210> 14
<211> 45
<212> DNA/RNA
<213> 236Fw(Unknown)
<400> 14
gattacttta gagtgggttg aaaagggaca ctcctctcat gatga 45
<210> 15
<211> 52
<212> DNA/RNA
<213> 236Ry(Unknown)
<400> 15
caacgtactc taaagtaatc atgtctttag tgtcttttaa gtgtctaacg tg 52
<210> 16
<211> 45
<212> DNA/RNA
<213> 236Fy(Unknown)
<400> 16
gattacttta gagtacgttg aaaagggaca ctcctctcat gatga 45
Claims (7)
1. A coupling enzyme YND mutant with high specificity of 3',5' -adenosine diphosphate based on molecular docking is characterized in that: the gene sequence of the YND mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the amino acid sequence of the YND mutant is SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
2. The molecular docking based highly specific coupling enzyme YND mutant of adenosine 3',5' -diphosphate according to claim 1, characterized in that: molecular docking software Vina1Used to design YND with high specificity for PAP substrate, the three-dimensional structure of YND was defined as PDB ID 1K9Y2The protein structure of (a); mutants of YND were constructed using a Discovery Studio Visualizer; both substrates PAP and PAPs were plotted using ChemDraw; in the butt-joint study, the lattice spacing was set to
The center (x, y, z) of the lattice point is (9.623, 16.925, -23.414), and the parameters of the lattice point are set as
The number of conformations obtained by docking was 20.
3. The molecular docking based highly specific coupling enzyme YND mutant of adenosine 3',5' -diphosphate according to claim 1, characterized in that: the YND mutant is induced and expressed by an escherichia coli expression system.
4. The molecularly-docked high-specificity coupling enzyme YND mutant of adenosine 3',5' -diphosphate (ADP) according to any one of claims 1 to 3, characterized in that: the YND mutant is stored in a storage solution, can maintain the activity for 3 months at the temperature of-80 ℃, and is diluted by a corresponding buffer system for use.
5. Use of the molecular docking based highly specific coupling enzyme YND mutant of adenosine 3',5' -diphosphate according to any of claims 1 to 4 for the determination of sulfuryl transferase.
6. The method for preparing the coupling enzyme YND mutant with high specificity based on the molecular docking of 3',5' -adenosine diphosphate as claimed in any one of claims 1 to 4, wherein: the method comprises the following steps:
firstly, designing a YND gene obtained by NCBI, inserting XhoI and NcoI enzyme cutting sites at two ends of the gene, checking and utilizing degeneracy of codons to remove the XhoI and NcoI enzyme cutting sites existing on the gene, wherein the modified amino acid sequence is SEQ ID NO.5, and the gene sequence is SEQ ID NO. 1;
synthesizing a modified YND gene by a gene artificial synthesis method, connecting the obtained YND gene with a PET28a (+) linear vector subjected to restriction endonuclease digestion by XhoI and NcoI by using solution I ligase to construct an expression vector for expressing YND;
obtaining mutant DNA by using an overlapping PCR method, designing a site-directed mutation PCR primer according to a site needing mutation, which is obtained by butting a YND sequence and molecules, selecting a homologous sequence containing a mutated base 15-25bp by taking a mutant site as a center, selecting a base 17-30bp upstream and downstream as a primer, and designing primer sequences from SEQ ID NO.7 to SEQ ID NO. 16;
transforming the constructed mutant plasmid into E coli JM109 competent cells for amplification and carrying out sequencing verification on the cells;
transforming the mutation plasmid with successful sequencing into E coli BL21,selecting transformed single colony, culturing in 5mLLB culture medium test tube overnight, inoculating the obtained bacterial liquid into 1L triangular flask liquid culture medium, liquid culturing at 37 deg.C to OD600When the temperature is up to 0.6, IPTG is added and the mixture is placed in a shaking table at the temperature of 16 ℃ for induction expression; collecting thalli after 20 h;
taking lysate suspension thalli, carrying out ultrasonic disruption on the thalli in ice bath at 12000rpm for 30min, and collecting supernatant;
purifying target protein by nickel column affinity chromatography, ultrafiltering protein obtained by nickel column affinity chromatography with stock solution, and replacing buffer system to obtain YND mutant, and storing at-80 deg.C;
wherein the stock solution is 50Mm Hepes buffer solution, and the pH value is 7.5.
7. The method for preparing the coupling enzyme YND mutant with high specificity based on the molecular docking 3',5' -diphosphonidine as claimed in claim 6, wherein: the YND is yeast 3',5' -diphosphatase,www.pdb.org；PDB:1KA1。
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| US20040110259A1 (en) * | 2001-08-03 | 2004-06-10 | Baugh Mariah R | Drug metabolizing enzymes |
| CN103451165A (en) * | 2013-08-06 | 2013-12-18 | 浙江师范大学 | 3'-Phosphoadenosine 5'-phosphate specific 3'-nucleotidase, and construction method and application thereof |
| CN108728477A (en) * | 2017-04-24 | 2018-11-02 | 华东理工大学 | A kind of efficient Transpositional mutation system and construction method |
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2021
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|---|---|---|---|---|
| US20040110259A1 (en) * | 2001-08-03 | 2004-06-10 | Baugh Mariah R | Drug metabolizing enzymes |
| CN103451165A (en) * | 2013-08-06 | 2013-12-18 | 浙江师范大学 | 3'-Phosphoadenosine 5'-phosphate specific 3'-nucleotidase, and construction method and application thereof |
| CN108728477A (en) * | 2017-04-24 | 2018-11-02 | 华东理工大学 | A kind of efficient Transpositional mutation system and construction method |
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