An in vivo cytochrome P450_cin (CYP176A1) catalytic system for metabolite production

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• 作者：Kate E. Slessor ; David B. Hawkes ; Anthony Farlow ; Andrew G. Pearson ; Jeanette E. Stok ; ^{j.stok1@uq.edu.au} ; James J. De Voss ; ^{j.devoss@uq.edu.au}
• 关键词：Cytochrome P450 ; In vivo ; Escherichia coli ; Cineole ; Camphor
• 刊名：Journal of Molecular Catalysis B ; Enzymatic
• 出版年：2012
• 期刊代码：55_13811177
• 类别：ce
• 出版时间：July, 2012
• 卷：79
• 期：Complete
• 页码：15-20
• 文件大小：389 K

摘要

Cytochrome P450_cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6尾-hydroxycineole. P450_cin uses two redox partners in vitro for catalysis: cindoxin, its physiological FMN-containing redox partner, and Escherichia coli flavodoxin reductase. Here we report the construction of a tricistronic plasmid that expresses P450_cin, cindoxin and E. coli flavodoxin reductase and a bicistronic plasmid that encodes only P450_cin and cindoxin. E. coli transformed with the bicistronic vector effectively catalysed the oxidation of 1,8-cineole, with the endogenous E. coli flavodoxin reductase presumably acting as the terminal electron transfer protein. This in vivo system was capable of producing enantiomerically pure (1R)-6尾-hydroxycineole in yields of 鈭? g/L culture, thus providing a simple, one-step synthesis of this compound. In addition, the metabolism of (1R)- and (1S)-camphor, structural homologues of 1,8-cineole was also evaluated in order to investigate the ability of this in vivo system to produce compounds for mechanistic studies. Significant quantities of five of the six possible secondary alcohols arising from methylene oxidation of both (1R)- and (1S)-camphor were isolated and structurally characterised. The similarity of the (1R)- and (1S)-camphor product profiles highlight the importance of the inherent reactivity of the substrate in determining the regiochemistry of oxidation in the absence of any specific enzyme-substrate binding interactions.