Modification Of Catalytic Activity And Substrate Selectivity Of 7α-Hydroxysteroid Dehydrogenase Based On Semi-Rational Design

Bear bile powder is a traditional rare and precious medicinal material,which is mainly obtained by artificial drainage of Ursus thibetanus.With the development of protein engineering and biotechnology,it is possible to cultivate bear bile powder in vitro.In the early stage,the research group established a technology for cultivation of bear bile powder by coupling of 7α-HSDH and 7β-HSDH to convert taurochenodeoxycholic acid(TCDCA)of poultry bile powder to tauroursodeoxycholic acid(TUDCA)in vitro.Due to the content of TCDCA in chicken bile powder is 42.58%and the non-bile acid components are similar to natural bear bile powder,it becomes the best natural substrate for biocatalytic cultivation of bear bile powder.However,chicken bile powder contains 4.743%of Taurocholic acid(TCA).The reported 7α-HSDHs are active on TCDCA and TCA at the same time,leading to the introduction of a new impurity tauroursocholic acid(TUCA)in the product.TUCA can cause colorectal cancer,increase the difficulty of separation and purification and affect the quality of the product.Therefore,screening or constructing a key catalytic enzyme with substrate selectivity and excellent enzymatic quality—7α-HSDHs is the key to the efficient large-scale cultivation of bear bile powder in vitro.Therefore,based on protein sequence information,secondary structure,and three-dimensional modeling analysis,this study applied genetic engineering technology to semi-rationally modified the new 7α-HSDH(St-2-2)obtained by the research group in the early stage.Mutants with significantly improved catalytic activity,thermal stability,and substrate selectivity were obtained.The role of key amino acid sites and the relationship between structure and function were discussed and analyzed.Further joint mutations were carried out to obtain a mutant I255G△C6 which maintains high activity on TCDCA but has no activity on TCA.It was applied to the catalytic conversion of chicken bile powder to prepare bear bile powder,and the transformation conditions were studied.It is the first time to obtain 7α-HSDH mutant with substrate selectivity for TCDCA and TCA,which provides a feasible strategy for the substrate selectivity modification of hydroxysteroid dehydrogenase,and provides a method for the catalytic conversion of chicken bile powder to prepare bear bile powder.The substrate-selective catalyst laid the foundation for the industrialization of bear bile powder in vitro.The main research contents and results are as following:(1)Based on the relationship between protein sequence-structure-function,the C-terminal of St-2-2 was modified by semi-rational design,and 6 mutants with 2-fold increase in catalytic activity and one substrate-selective mutant St-2-2△C10 were obtained.1)The catalytic activity study showed that the activity of mutants I255F,I255Q,I255A,I255N,I255S,and I255G on TCDCA increased by 109.5%,116.1%,137.2%,233.1%,236.4%,and 285.9%,respectively;The activity of TCA was reduced to 4.9%,15.5%,5.8%,7.3%,7.2%,and 4.6%of the wild type respectively,realizing the reversal of catalytic activity.2)The C-terminal truncation strategy was used to further explore the effect of C-terminal on the catalytic activity of the enzyme.It was found that the mutant St-2-2△C10 completely lost the activity on TCA and sodium glycocholate hydrate(GCA),while still retaining the catalytic activity of 14.9 U/mg on TCDCA.It appears substrate selectivity.3)I255A,I255G,St-2-2△C10 were selected for thermal stability study.The results showed that the Tm of I255A and I255G were increased by3.2~oC and 4.1~oC compared with the wild type,while the Tm of St-2-2△C10 was 4.0~oC lower than that of the wild type;MD analysis at 310 K also found that the RMSD of I255A and I255G was more stable than that of the wild type,while the RMSD of St-2-2△C10 was significantly higher than that of the wild type.4)The three-dimensional structure analysis results showed that the C-terminal amino acid residues are involved in the interactions within and between subunits.Positions 255 of I255A and I255G form new hydrogen bonds with Ser253 to increase thermal stability,while the tail in St-2-2△C10 is shortened,the number of interacting hydrogen bonds decreases,leading to a decrease in stability.The above research results indicate that the interaction between the C-terminal amino acid residues plays an important role in maintaining the catalytic function and thermal stability of St-2-2,and the modification of the C-terminal is a feasible strategy to achieve substrate selectivity enzymes.(2)Based on molecular docking technology,changing the coenzyme binding site residues at N-terminal by semi-rational modification to obtain multiple mutants with increased activity.1)The catalytic activity study showed that activity of T15A on TCDCA increased by 685.4%(p<0.001);the catalytic activity of T15L,T15Q,T15R,T15G,R16S,R16G,R16A,and R16Q on TCDCA increased by 131.2%,173.6%,180.6%,217.8%,106.4%,106.8%,150.2%,and 335.4%,respectively.On the contrary,the catalytic activity on TCA decreased to 16.4%,40.3%,41.6%,48.8%,22.0%,8.0%,39.9%,and 28.9%of the wild type.2)T15A,R16A,and R16Q with greatly improved catalytic activity were selected for thermal stability study.The results showed that their Tm values were increased by 4.2~oC,6.0~oC,and 7.0~oC respectively;MD analysis at 310K also found that the RMSD of the mutant was higher than that of the wild type.3)The secondary structure determination by circular dichroism spectroscopy showed that in T15A,R16A,and R16Q the content ofα-helix decreases,and the content ofβ-sheet increases to maintain the stability of the protein structure;the amino acid hydrophilicity and hydrophobicity analysis results showed that the amino acid located inside the molecule.Mutation of amino acids(Thr15,Arg16)into more hydrophobic amino acids(Ala,Gln)can improve thermal stability.4)The results of homology modeling and three-dimensional structure analysis showed that Arg38 in T15A and Asp194 in R16Q increased the anchoring effect with the coenzyme,thereby strengthening the tightness of the connection between the coenzyme and the enzyme and making the configuration of the substrate in the binding pocket more stable and improving the catalytic activity of the enzyme.The above results indicate that changing the amino acid residues of the N-terminal is a feasible strategy to improve the catalytic activity and thermal stability of HSDHs.(3)I255G△C6 with substrate selectivity was obtained based on multiple modification strategies,and further used in the catalytic conversion of complex substrates to prepare bear bile powder.1)I255G△C6 has no catalytic activity on TCA,but its catalytic activity on TCDCA still remains 40.3±6.1 U/mg,which is 2.7 times that of St-2-2△C10.Thermal stability studies showed that the Tm of I255G△C6 is59.7~oC,which is 3.1~oC higher than St-2-2;MD analysis at 310 K found that the RMSD value of I255G△C6 fluctuated between 1.5-2.0(?).I255G△C6 is more stable than the wild type;further structural analysis showed that the key site I255G was retained,and the force with Ser253 was added to increase its thermal stability.3)Using I255G△C6combined with 7β-HSDH to catalyze the conversion of chicken bile powder,and optimize the reaction conditions.The results showed that the best p H was 9.5,the best substrate concentration was 2 m M,the best coenzyme concentration was 2 m M,the best reaction time was 5 h,and the best buffer concentration was 50 m M Gly-Na OH.According to HPLC-ELSD analysis,I255G△C6 does not catalyze the conversion of TCA to TUCA.It can be seen that using I255G△C6 with substrate selectivity as a catalyst,the catalytic conversion of chicken bile powder not only avoids the introduction of new impurities,but also retains non-bile acid components,laying a foundation for in-situ biotransformation of bear bile powder in vitro.