Computer-aided Design Of Electron Transport Chain And Its Effect On Vitamin D₃ Hydroxylase Vdh Activity

Vitamin D₃ hydroxylase(Vdh)is a cytochrome P450 monooxygenase from Pseudonocardia autotrophicais that catalyzes the hydroxylation of vitamin D₃(VD₃)to produce the active forms of VD₃—25 hydroxyvitamin D₃(25(OH)VD₃)and 1?,25-dihydroxyvitamin D₃(1?,25(OH)2VD₃).The preparation of active VD₃ using Vdh has the advantages of green and good stereoselectivity,and has a better commercial prospect than chemical synthesis.Vdh needs to be combined with redox partner to catalyze the hydroxylation reaction.However due to lack of research on the natural redox partner of Vdh,and the commercial electron transport chain cannot be well coupled with Vdh.It is difficult to meet people's catalytic needs.At the same time,reports on the characteristics of the catalytic substrate of Vdh are very limited,and further study is needed.Therefore,this study used homology modeling,virtual alanine scanning and molecular dynamics calculation methods to research the of Vdh electron transfer chain and virtually screens and validates the substrate spectrum of Vdh.The detailed contents are as follows:(1)Rational design of VD₃ hydroxylase key sites based on molecular dynamics simulations and study of effect on VD₃ conversion efficiency.In this study,the structure of spinach ferredoxin(Fdx)was obtained by homology modeling,and the complex structure of Vdh-Fdx was obtained by molecular docking.This was followed by a saturation virtual mutation of key residues(Arg55,Arg99,Lys100,Gly103,Arg104,Thr107,Val108,Arg109,Phe339,Phe340,Phe346,Gln351,Arg354 and Arg358)using an alanine scan to find the acting residues at the interface of Vdh and Fdx binding.The mutants Vdh T107K,Vdh V108W were selected as the best mutants according to the mutation energy(Vdh T107K:-3.31 kcal/mol,Vdh V108W:-2.27 kcal/mol).Using site-directed mutagenesis,the Vdh T107K and Vdh V108W genes were obtained,and they and wild-type Vdh were ligated with p MD19-T and p ET-28a,successively.The target protein was then obtained by in vitro induced expression.The expressed wild-type Vdh and Vdh mutant proteins were used to catalyze VD₃ respectively.The catalytic products were analyzed by high performance liquid chromatography(HPLC).The results showed that the average conversion efficiency of the mutant protein was higher than that of wild-type Vdh,with 28.80%for mutant protein Vdh T107K and 25.72%for Vdh V108W,compared with 9.12%for wild-type Vdh.Finally,the reasons affecting the transformation efficiency of mutant protein VD₃ were explored by computer-assisted techniques.(2)Effect of linker flexibility on the conversion efficiency of fusion proteins of VD₃hydroxylase and cytochrome P450 reductase.The recombinant cloning plasmids of fusion proteins Vdh-G-CPR(rigid linker peptide)and Vdh-P-CPR(flexible linker peptide)were constructed by adding different flexible linker peptides between Vdh and cytochrome P450reductase(CPR)by PCR amplification.Obtaining target proteins by in vitro induction.Finally,the molecular dynamics of the rigid linker peptide and the flexible linker peptide were simulated by 100 ns.It was found that the flexible linker peptide underwent irregular curling during the simulation,while the rigid linker peptide maintained a linear structure throughout.Among them,the fusion protein Vdh-P-CPR had the highest average conversion rate of 61.31%for VD₃,and the average conversion rate of Vdh-G-CPR for VD₃ was 30.38%(3)Screening of potential substrate spectrum of Vdh based on molecular dynamics simulations.The ZINC compound database was screened using molecular docking,combined with scoring,property screening and its generalized Bern surface area method(MM/GBSA)to calculate the binding free energy,from which four small molecules were finally selected for experimental validation.From the experimental results,the experimental results are consistent with the screening results,selected four small molecules can react with Vdh,and van der Waals forces are the main driving force for the binding of Vdh to small molecules.Summarizing the properties of the small molecules it was concluded that the compounds selected for the database have high similarity to VD₃ and all have the possibility of active sites where hydroxylation occurs.Presumably,Vdh is a potential biocatalyst for the production of steroidal drugs.