Comparative Analysis Of Three Pairs Of Surrogate Redox Partners For In Vitro Activity Reconstitution Of Class Ⅰ Cytochrome P450 Enzymes

Cytochrome P450 enzymes(cytochrome P450 enzymes,P450s)are a class of hemecontaining protein superfamily,with extensive catalytic activity and unique structural characteristics.As one of the most versatile biocatalysts in nature,P450s play crucial roles in the biosynthesis of steroids,the biodegradation of xenobioticsin and various natural product bioproduction,by selectively oxidazing C-H bonds,N-H bonds,S-H,etc.In the field of synthetic chemistry,P450s are very attractive to product fine chemicals and drugs due to that can catalyze regio-and stereoselective oxidations of inert C-H bonds in complex organic molecular scaffolds.The most abundant Class I cytochrome P450s,including prokaryotic and mitochondrial P450s,which usually require a couple of redox partner(RP)proteins composed of flavincontaining ferredoxin reductase(ferredoxin reductase,FdR)and iron-sulfur cluster-containing ferredoxin(ferredoxin,Fdx).The redox partner system is responsible for the sequential shuttling of two electrons from the cofactor NAD(P)H to the heme of P450 along the electron transfer chain "NAD(P)H→FdR→Fdx" to activate O2 for further substrate oxidation.Although numerous P450 genes have been annotated,their innate pairs of RP are normally difficult to identify due to their biological variations and/or difficulty to express.The RP systems are widely found in bacteria and the mitochondria in plants and animals,however,the genes encoding P450 and their potential RP are often located far away from each other.Thus,since the identification of cognate redox partners for bacterial P450s is often impossible,characterized heterologous redox partners are usually used instead to support reactions catalyzed by P450s.Spinach ferredoxin and ferredoxin reductase have commonly used as the surrogate redox partner,which are expensive and difficult to industrialize.With the development of sequencing technology,more and more RP genes have been excavated,but how to quickly select a suitable RP is still a difficult problem.Different pairs of RP may change the site selection and type of P450 enzymes reactions,and generate new catalytic product.Therefore,appropriate RP plays a crucial role in the reconstruction of P450 activity.This study comprehensively explored three surrogate redox partners,including Adx/AdR from bovine,Pdx/PdR from Pseudomonas putida,and SelFdx1499/SelFdR0978 from Synechococcus elongatus PCC 7942,to reconstruct the function of three selected bacterial P450s with industrial application value.Three P450s were selected to accept electrons delivered by electron transport chain,including PikC(CYP107L1),P450sca-2(CYP105A3)and CYP-sb21(CYP107Z14),which are responsible for production of macrolide antibiotics,the cholesterol-lowering drug pravastatin,and a hair-growth-stimulating agent γ-hydroxy-N-methyl-l-Leu4-CsA(CsA-4-OH).Three P450s are involved in the biosynthesis of microbial natural products,and their endogenous RP has not been reported.Through studying the kinetics of FdR,calculating the electron transfer efficiency of different RP combinations,and analyzing the P450-Fdx and FdR-Fdx protein complex by protein molecular docking(distance between catalytic centers,contact area,interaction of surface key amino acids,etc.),it shown that SelFdx1499/SelFdR0978 was the best alternative RP among the three RP pairs.This was consistent with the experimental data of all possible 27 combinations(3 FdR×3 Fdx×3 P450)which were tested to determinethe product distribution/ratio of each reactions.These studies discovered that SelFdx1499/SelFdR0978 was the most effective surrogate RP to support all three P450 reactions.This study identified a series of systematic biochemical experimental methods,which could quickly select suitable surrogate RP for specific Class I P450 enzymes and provide a method reference for future research on RP system of P450.Meanwhile,SelFdx1499/SelFdR0978 with high electron transfer efficiency is expected to become a general RP for the functional study of P450 or the reconstruction of P450 activity in vitro with industrial application value,prior to identification of their endogenous electron donors.At the same time,this work utilizes the recently available Alphafold2 technology,which has important guiding significance for the future study of P450 enzymology.