Controlled oxidation of aliphatic C-H bonds in metallo-monooxygenases: Mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons by Cytochrome P450 BM3

• Main Authors: Chung-Ling Yang, 楊宗霖
• Other Authors: Sheng-Fa Yu
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/t2uwv5

博士 === 國立臺灣科技大學 === 應用科技研究所 === 103 === The control oxidation of aliphatic C-H bonds in regio- and/or stereo-selective manner by metalloenzymes is of great interest to scientists. Cytochrome P450 BM3 from Bacillus megaterium oxidizes C12-C20 fatty acids at the ω-1, ω-2, or ω-3 position of the C?{H bond, respectively. We employ the molecular manipulation techniques to overexpress the recombinant BM3 strain and carry out its site-directed mutagenesis study for generation of a variety of strains towards variable selective oxidation with different substrates. In my study, strain 3mt, A74G F87V L188Q is exploited for the controlled oxidations of medium-chain length alkanes. Single mutation variant, BM3 mutant Ala328Phe (F328), was found with its capability of selective oxidation at the ω?{1 position of C4-C10 straight-chain alkanes. Interestingly, long-chain fatty acids (C12-C20) are no longer its substrates. It provides a great base to allow us engineering P450 BM3 protein from normal alkanes with medium-chain length such as n-octane gradually to smaller alkane such as n-butane for subsequent studies of controlled oxidation. The introduction of the second mutation, at Leu188Pro (P450 BM3-FP188) can improve the catalytic efficiency from butane to 2-butanol for 12.5%. In addtion, introducinge Ala74Glu (P450 BM3-FPE74) can shrink the substrate pocket down to half of the volume and significantly enhance the catalytic activity of butane to 2- butanol for nearly 40%. Typically, enzymes invoke host–guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. To further investigate this issue, we also develope a series of deuterated and fluorinated aliphatic substrates as probes to gain insights into the controlled C-H oxidations of hydrocarbons facilitated by these enzymes. The effects resulting from the introduction of deuterated butane (isotopomers) and fluorinated octane (Bioisostere) substituents on the mechanistic insights for C-H oxidation and the controls for regio-specificity and stereo-selectivity of the C-H bond activation are discussed.