Research On The Mechanism Of Bioconversion And Catalyzation By P450(CYP105D7) From Strentomyces Avermitilis

Cytochrome P450 of Streptomyces avermitilis are important catalysts for the biosynthesis and oxidation of xenobiotic chemicals in bacteria, such enzyme has attracted wide attention of the chemists and biologists due to their regio- and stereospecificity during the catalysis of substrates. As one of the enzyme family members, CYP105D7 is able to catalyze the monooxygenation of a variety of natural products into novel compounds. It is known that flavonones play positive roles in the treatment on breast cancer, and mevastatin is a kind of high efficient anticholesteremic agent, moreover, studies have indicated that the activities will all be enhanced when introducing hydroxyl groups into these compounds. In this thesis, the biotransfonnation and catalytic mechanism of flavonone naringenin and pinocembrin as well as mevastatin(a statin chemical)by CYP105D7 were investigated. By means of heterologous expression and purification of CYP105D7 and determination with UV spectroscopy, the binding affinity of enzyme with substrates were detected and determined, meanwhile, bioconversion in vitro and whole cell biotransformation in vivo of these compounds were carried out, trying to obtain the hydroxylated products with higher activities. The molecular dockings of CYP105D7 with these compounds were also conducted to analyze the catalytic reaction mechanism of the enzyme. The calculated results of UV difference spectroscopy indicated that the dissociation constants and Hill coefficients of naringenin and pinocembrin were Kd= 103±3μM, nH=1.25±0.03 and Kd= 52.3±2.1μM,nH= 1.47±0.06, respectively. HPLC and LC-MS analysis demonstrated that CYP105D7 had the ability to hydroxylate both naringeinin and pinocembrin, and the bioconversion product of naringenin was proved to be eriodictyol at the 3’site, moreover, the substrate-binding pocket of CYP105D7 was sufficiently wide to accommodate two naringenin molecules simultaneously. And multiple naringenin molecules are bound to CYP105D7 with weak cooperativity, which may be necessary to the 3’-hydroxylation of flavanone. For the bioconversion of mevastatin, the UV difference spectroscopy and molecular docking experiments showed that the possibility of substrates biotransfonnation by CYP105D7 was very high, and the subsequent investigation found that bioconversion of mevastatin was affected by the lactone and acid forms of this compound.