The Study of Regio- and Stereo-selective Hydroxylation of Butane via Cytochrome P450 BM3 Variants

• Main Authors: Lin, Cheng-hung, 林承鴻
• Other Authors: Yu, Steve Sheng-Fa
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/22999881363337852555

碩士 === 國立中正大學 === 化學暨生物化學研究所 === 99 === Cytochrome P450 BM3 from Bacillus megaterium display a rather excellent substrate specificity. This enzyme can only hydroxylate fatty acid by using atmospheric dioxygen as an oxidant. We successfully expressed and purified the recombinant His-tagged protein (P450 BM3) from E. coli. by a simple metal-affinity chromatography. Employing site-directed mutagenesis studies, we obtained the three strains to study their butane activation chemistry without any alternation of P450 reductase. The three mutants are cytochrome P-450 BM3 Ala328Phe denoted as “F328”, P-450 BM3 Ala328Phe and Leu188Pro that is denoted as “F328+P188”, and cytochrome P-450 BM3 Ala328Phe,Leu188Pro,Ala74Glu that is denoted as “F328+P188+E74”. These three strains exhibiting iron heme could carry out n-butane activation to 2-butanol in catalytic manner. Interestingly, n-butane can not be oxidized by the wild-type enzyme. The turnover activity data indicates that the best mutant for butane activation is F328+P188+E74. Using chiral di-deuteriated n-butanes as its subtrates, to analyze the distribution of products by GC and GC-MS, we can derive the equilibrium constants for secondary C-HR versus C-HS pointing to the iron heme center and the H/D kinetic isotope effects on the high valent iron oxo species (compound I) in P450 enzyme across the C-H bond. The outcomes on the hydroxylation mediated by cytochrome P450 BM3 variants presented as fully retention of configuration as well as the H/D kinetics isotope effects in the range of 3.45.5 suggest the reaction proceeds in concerted insertion manner. Evidently, when the small alkanes enter the pocket of enzymatic reactive center, there are significant weak interactions including van der Waals interaction or London Dispersive force to tune the products hydroxylation with high regio-selectivity and stereo-selectivity.