Molecular Evolution Of P450_LaMO Monooxygenase For Enhancing Its Oxyfunctionalization In Tetrahydronaphthalene And Its Derivatives

Cytochrome P450 monooxygenase is a type of oxidase for hydroxylating inert C-H bonds and widely distributed in nature.Self-sufficient P450 monooxygenases from CYP116B subfamily has the novel FMN and Fe2S2 domains,which is different from typical representative P450BM3.It is worth noting that the substrate specificity and the improvement of oxyfunctionalization of CYP116B monooxygenases had not been performed systematically exploration.Based on the spectrum characterization of CYP116B monooxygenases,though it showed obvious activity for tetralin,the coupling efficiency,thermostability and selectivity were still low.With the development of molecular evolution,it provides the strong guarantee for improving the catalytic performance of P450 monooxygenase.In this work,three CYP116B monooxygenases were measured the demethylation activity for a series of O-methylated substrates and then profiled their fingerprints.It is showed that these P450s preferred the substrates of alkylbenzene and its derivatives.Moreover,the(S)-selective hydroxylated products tetralol also was an important building block in drug synthesis.Thus,in order to obtain optical active tetralol,we performed the molecular evolution for better oxyfunctionalization of P450LaMO contained activity,electron coupling efficiency,thermostability and selectivity.The whole work could be divided into three sections,more details are as follows.Section ?:Substrate specificity analysis of CYP116B monooxygenases.We firstly designed and synthesized four types of O-methylated substrates as probes,including monoterpenes,cycloalkanes,aromatic compounds and steroids.The Purpald dye-based colorimetric assay was used to measure demethylation activity of three member monooxygeases P450RpMO,P450ArMO and P450CtMO for the synthesized probe substrates.Meanwhile,the reliability and viability of this method were investigated,and the results showed that within the linearity concentration range of 2-100 ?M,the standard addition was about 84%to 104%.Furthermore,the intermethod comparison between chromatography and colorimetric assay achieved the positive correlation.Based on these results,we featured the demethylation activity-based fingerprint of these three P450s.The results showed that these P450s preferentially oxidized substrates with the core structure 1,2,3,4-tertahydronaphthalene.Section ?:Molecular evolution to improve the activity,electron coupling efficiency and thermostability of P450LaMO.The catalytic performance of wildtype P450LaMO is severely hindered by a low reaction rate,poor electron coupling efficiency(CE)and fragile thermostability.Herein,a simple transdomain combination mutagenesis(TDCM)strategy was proposed for this multi-domain protein.After focused mutagenesis on the heme domain,a triple mutant H3(N119C/V264A/V437G)was hit,that improved the turnover frequency(TOF)and CE of P450LaMO by about 7.8-fold and 3.0-fold,respectively.A redox domain-based mutant with higher cytochrome c reduction activity,MR1(M612L/K774Y),mediated more efficient electron transfer,elevated the TOF by 4.9-fold,and the coupling efficiency by 4.2-fold.The beneficial effect was further enhanced by combining the mutation sites from different domains,resulting in a combinatorial mutant(N119C/V264A/V437G/M612L/N694D)with a 9.1-fold increase in coupling efficiency,10-fold in TOF,as well as+3.8? in thermostability(T5010).Meanwhile,for series of tetrahydronaphthalene derivatives,this combinator showed better hydroxylation activity.This work suggested that this TDCM strategy is efficient to improve the performance of CYP116B subfamily members.Section ?:Based on the characterization results of wild type,we found that P450LaMO catalyzed core structure 1,2,3,4-tetrahydronaphthalene to less reported(S)-tetralol but with low enantioselectivity(e.r.=66:34)and product selectivity(alcohol:ketone(a.k.)=76:24).We firstly performed the Phe scan for five residues(L97,T121,V123,N124 and A275)lining the acitve pocket.The resulting mutant RJ1(T121F)showed an improved e.r.(93:7)and a.k.(92:8).Through iterative saturation mutagenesis based on RJ1,we obtained RJ2(T121F/Y385F/M391L)with e.r:of 95:5 and a.k.of 95:5.After polarity optimization of site T121 from RJ2,the e.r.,a.k.of RJ3(T121V/Y385F/M391L)were up to 98:2,99:1,respectively.The TOF and TTN of RJ3 was 0.71 min-1,1360,about 18-fold and 12-fold improvement compared to wild type.Moreover,the improved TOF and TTN of mutant RJ33(T121P/Y385F/M391L)was about 21-fold,18-fold,respectively.But the enantioselectivity decreased slightly.Meanwhile,the mutant RJ6(E282V/T283Y/T121F)achieved the complementary(R)-selectivity(e.r.=23:77)but the product selectivity was just a.k.=76:24.The results of molecular docking showed that the enhanced enantioselectivity and activity was mainly due to the improved hydrophobic interaction based on sites V121,F385 and L391.Furthermore,the enviromantal factors such as the cosolvent,temperature,pH were also optimized for improving the product selectivity.Therefore,we used the RJ33 to perform the biotransformation of alkylbenzene derivatives in 10-mL reaction.The biocatalyst could produce the alkylbenzene alcohol with high(S)-enantioselectivity and product selectivity.In conclusions,we characterized CYP116B members based on the analysis of activity-based fingerprint.To solve the problems of low coupling efficiency,thermostability and selectivity of P450LaMO,we performed the transdomain combination mutagenesis and mutation scan to improve the activity and enantioselectivity,respectively.Finally,the mutant could efficiently transform the alkylbenzene compounds with better activity,high enantioselectivity and product selectivity.