Studies On Functional Modulation Effects Of Redox Partners Toward The Cytochrome P450 Monooxgenase MycG

Cytochrome P450 monooxygenases,a class of heme b-dependent protein superfamily that participate in more than 20 different types of chemical reactions,such as hydroxylation,epoxidization,dealkylation,deamine and so on.P450s paricipatiate in a huge number of physiological reactions,including the biosynthesis of natural products and signaling molecules,human drug metabolism and the biodegradation of some heterologous substances,are considered as the most versatile biocatalysts in Nature.Most P450s can not work alone because their active form is generated by the two electrons from cofactor,such as NADPH or NAPH.Thus,redox partners that responsible for the electron transferring are usually indispensable for P450 catalytic reactions.During the long evolution process,various P450redox partner systems have been constituted by protein fusion and recombination.In order to functional reconstruction of P450s,the selection and combination of redox partner of specific P450 enzyme are often not only the key factors causing the failure or inefficient catalytic activity of P450 enzymes,but also are able to influence and change the product distribution of P450 enzymes.To date,no comprohensive study on the mechanism of how the combination and interaction between different redox partners and P450 enzymes in affecting the reaction types,catalytic efficiency and product distribution of P450s.In this study,the adaptation and interaction mode of the multifunctional P450 monooxygeanse MycG(CYP107E1)from the rare actinobacteria Micromonospora griseorubida and the redox partner RhFRED from Rhodococcus sp.NCIMB 9784 were systematically investigated by constructing various fusion and separated proteins.Specifically,the two domains of RhFRED were artificially separated and heterogeneous expressed as two stand-alone FMN and Fe2S2 proteins to mimic the two components system of redox partner.On the other hand,the protein organization arichitecture of these two domains was exchanged,or shuffled,and then engineered in a single polypeptide chain with MycG into artificial "self-sufficient" P450s fusions.In vitro biochemical reactions were carried out to explore the effects of engineered different redox partner combinations toward the MycG-catalyzed bioconversion of mycinamicin-Ⅳ(M-Ⅳ)and the variations of electron transfer efficiency.As results,among the 20 engineered P450 catalytic systems,the function of MycG were successfully reconstituted in 12 combinations by delivering the three oxidative products(M-Ⅰ,M-Ⅱ and M-Ⅴ),and one demethylation product(dMe-M-Ⅳ).4 combinations only led to the production of M-Ⅰ,M-Ⅱ and M-Ⅴ.In addition,no detectable products were found in the other 4 combinations.In conclusion,by simulating natural evolutionary strategy,the effects of three different catalytic systems of redox partner and P450,together with different organization types on functional modulating effects of MycG were performed systematically.These results indicated that the P450 catalytic properties are affected and modulated not only by the selectivity and combination of redox partner,but also by the domain position and organization of redox partner to P450s.Unlike the traditional directed evolution of P450 which engineering the amino acids of protein,this study demonstrated redox partner engineering is an effective engineering strategy that will facilitate the development of optimal catalytic systems cytochrome P450 enzymes and redox partner for specific chemical reactions.