Study On The Structural Mechanism Of Redox Partners Regulating The Catalytic Activity Of P450 Enzymes

Cytochrome P450 enzymes(abbreviated as P450 or CYP)are a class of monooxygenases containing ferric hemoglobin widely found in nature.P450 can perform site-and stereospecific oxidation of various complex organic compounds under mild conditions,playing an important role in natural product biosynthesis,heterologous metabolism,and industrial catalysis,and are a class of versatile biocatalysts.Due to their significant applications in environmental protection,chemical industry,medicine,and other fields,the research on P450 has gradually become a hotspot in the field of biocatalysis in recent years.To activate molecular oxygen,Type Ⅰ P450 enzymes require the orderly transfer of two electrons from NAD(P)H to the active center of P450 enzymes via redox partners.In this process,the dual-electron carrier ferredoxin reductase(FdR)receives two electrons from NAD(P)H and sequentially reduces two equivalent amounts of ferredoxin(Fdx),and the singleelectron carrier Fdx interacts directly with P450 and transfers the two electrons in turn.Therefore,Fdx and FdR are important factors affecting the catalytic efficiency of P450 as the electron shuttle carriers necessary for the catalytic function of Type Ⅰ P450 enzymes.With the advent of the post-genome era and the rapid development of whole-genome sequencing technology,more and more redox partner genes have been found and characterized.which can help the discovery of efficient redox partner systems that enhance the catalytic efficiency of P450 enzymes.However,it is unclear how different redox partner combinations affect P450 activity,and the key mechanisms affecting electron transfer efficiency still need to be further explored,especially in the Type Ⅰ P450 enzyme system in which FdR and Fdx are independent electron transfer proteins.This thesis systematically studied this scientific question using the niques of structural biology and in vitro enzymatic reaction reconstruction,combined with other biochemical experiments.The first part is about the structural mechanism of redox partners affecting electron transport.The crystal structures of FdR0978 from Synechococcus elongatus and FdR2690 and FdR2141 from Streptomyces venezuela were solved,respectively.Compared with the structures of the other two FdRs,FdR0978 possesses an open-state interface for the prosthetic group FAD that plays a key role in electron transport,facilitating its proximity to the iron-sulfur cluster of Fdxs to complete efficient electron transport.The second part is the mechanism verification of FdRs on the catalytic efficiency of P450 enzymes.Firstly,a suitable ferredoxin Fdx1499 was identified by in vitro enzymatic remodeling using a random combination of redox partners and P450sca2.Then,under the condition of different FdRs,P450sca2 hydroxylated mevastatin to produce the cholesterol-lowering drug pravastatin,HPLC results showed that the combination of full-length and truncated FdR0978 had the highest substrate conversion rates(94.9%and 96.4%),while the combination of FdR2690 and FdR2141 could only achieve 9.0%and 26.9%.These results proved the important role of the FdR’s special structure in electron transport.On this basis,a fusion redox partner Fdx1499-FdR0978 with a flexible linker was constructed and screened,which can facilitate the efficient transformation of mevastatin by P450sca2.The third part describes the crystal structure of the P450 hydroxylase CYP-PA1 of Pseudonocardia autotrophica.The structure of its homologous protein CYP-sb21 was previously solved and CYP-sb21 was also modified by rational design.However,production of the by-product CsA-9-OH could not be decreased,hindering the efficient production of CsA4-OH,an efficient hair growth promoter.This section analyzes the crystal structure of CYPPA1,which also has a site-selective preference for CsA.Through structure comparison and sequence analysis,we found several possible key amino acids with a catalytic preference for generating CsA-9-OH.In summary,the crystal structures of three FdRs(2690,2141,0978)were resolved,and the possible structural mechanism of FdRs affecting the catalytic efficiency of P450 was verified in vitro enzymatic reconstitution.An Fdx1499-FdR0978 fusion protein was identified to achieve efficient transformation of mevastatin by P450sca2.Moreover,the crystal structure of CYP-PA1 was resolved,which may pave a theoretical foundation for the subsequent directed evolution of CYP-PA1 and CYP-sb21.