Study On The Cytochrome P450 Enzyme PikC_D50N As A Biocatalyst To Produce Human Drug Metabolites

Drugs can be metabolized by different subtypes of cytochrome P450 enzymes in the human liver to produce a variety of human drug metabolites(HDMs),thereby changing the activity,toxicity,stability,solubility and bioavailability of the parent drugs.Rigorous toxicological,pharmacological and pharmacodynamic testing of drug metabolites have become an essential part during modern drug development.Therefore,drug metabolites are important chemical standards in drug research.Due to the high region-and(or)stereoselectivity of P450 enzyme catalyzed oxidative reactions,preparation of HDMs by organic synthesis is suffering from the poor selectivity,low yield,high-cost and environmentunfriendly issues.In addition,human or mammalian P450 enzymes are often unstable membrane proteins,which limit their expression and application in heterologous hosts.Thus,development of biocatalysts with human P450-like function would become an effective way to solve this problem.The cytochrome P450 enzyme PikC is a monooxygenase derived from Streptomyces venezuelae involved in the biosynthesis of pikromycin and can be highly expressed in E.coli and other heterogenous hosts.Inspired by the special mutual recognition-mechanism of PikCsubstrates,this study developed the mutant PikCD50N into a potential biocatalyst for the preparation of HDMs,with a combination of methods including the substrate screening,in vitro enzymatic reaction,product identification,redox partners and electron donor study,protein structure analysis and catalytic mechanism speculation.The main research progresses of this thesis are as follows:1.Based on the enzymatic mechanism of PikC,19 clinical drugs containing N-alkyl groups were selected for in vitro enzyme reaction with PikCD50N,a high activity mutant of PikC.The results showed PikCD50N can convert 15 of them into various of products.Structure identification and product comparison revealed that PikCD50N takes a similar product profile with human P450 enzymes.2.Using the "PikCD50N-chlorpromazine as a model reaction,the effects of different redox partners and electron donor on product distribution were explored.Reaction amplification proved the application potential of PikCD50N as a biocatalyst.Docking experiments showed that the substrate chlorpromazine can be combined to the catalytic pocket of P450 enzyme in various catalytic conformations,thus illustrating the reason why PikCD50N can mediate multitype reactions.Through protein catalytic pocket comparison found that PikCD50N shares a similar catalytic pocket shape with the major human P450 enzyme CYP 3A4 involved in drug metabolism,preliminarily explaining similar metabolite profiles between them.In conclusion,this study firstly found the bacterial biosynthetic P450 enzyme PikCD50N has the catalytic function of human-like P450 enzyme,and PikCD50N has the advantages of strong plasticity,low-cost,high catalytic efficiency and wide range of substrates,which has the potential of practical application in the drug industry.This study also provides beneficial reference for the development of PikC and other biosynthetic P450 enzymes in the future.