Research On The Compactin Biotransformation System Mediated By A Cytochrome P450 Enzyme And The P450 Catalytic Mechanism

Compactin and pravastatin are competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase,which is an enzyme in the biosynthesis of cholesterol.Both of these compounds belong to the statin drugs which can be used to reduce cholesterol in humans.Pravastatin,which is a hydroxylated derivative of compactin,shows superior pharmacokinetic characteristics.So far,pravastatin is mainly composed of chemical synthesis by compactin.However,with the shortcomings of low productivity,environmental pollution and high cost of chemical synthesis,biotransformation has obtained more attention because of high efficiency,green environmental protection and high stereoselectivity.In this study,the bacterial cytochroime P450,CYP105D7,from Streptomyces avermitilis MA4680 was used as a biological catalyst to mediate compactin.CYP105D7 can be used in the presence of O2 and NAD(P)H as the oxygen and electron donor,respectively.An extensive screening process was used to determine the optimal transformation conditions for the catalytic hydroxylation of compactin at C6 position in vitro in the presence of three redox partners(Pdx/Pdr,seFdx/.seFdR and RhFRED).The three conditions afforded conversion rates of 6.3,1.8 and 14.7%,respectively.We also conducted a detailed analysis of the catalytic mechanism of CYP105D7 based on the enzyme kinetics of the Pdx/Pdr-mediated process,binding affinity and molecular docking analyses.These results showed that it was a single molecular combination method and had good binding affinities to the substrate.In order to improve biological efficiency,the optimal redox partner was subsequently used to construct a highly efficient electron transfer system.RhFRED was selected as the reductase domain and subsequently constructed the fusion protein CYP105D7-RhFRED using a combination of genetic engineering and enzyme engineering with a one-step cloning method.This construct shortens the distance between the P450 and the redox partner,leading to considerable improvements in the electron transfer and catalytic activity of this system.This study provides new biotransformation approaches and ideas for the production and productivity of pravastatin,and also investigates the CYP105D7-mediated hydroxylation of compactin and developed various optimized biotransformation processes.These results will not only offer a theoretical basis and feasibility,but will also provide a platform for the industrial application of CYP105D7 used as a biological catalyst and future research.