An in vivo cytochrome P450cin (CYP176A1) catalytic system for metabolite production

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Title An in vivo cytochrome P450cin (CYP176A1) catalytic system for metabolite production
Author Slessor, Kate E.; Hawkes, David B.; Farlow, Anthony; Pearson, Andrew; Stok, Jeanette E.; Voss, James J. De
Journal Name Journal of Molecular Catalysis B: Enzymatic
Editor Y. Asano, J.D. Stewart, F. Hollmann
Year Published 2012
Place of publication Netherlands
Publisher Elsevier
Abstract Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6-hydroxycineole. P450cin 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 P450cin, cindoxin and E. coli flavodoxin reductase and a bicistronic plasmid that encodes only P450cin 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-hydroxycineole in yields of ∼1 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.
Peer Reviewed Yes
Published Yes
Alternative URI http://dx.doi.org/10.1016/j.molcatb.2012.03.007
Copyright Statement Copyright 2012 Elsevier. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.
Volume 79
Page from 15
Page to 20
ISSN 1381-1177
Date Accessioned 2012-08-02
Date Available 2013-06-11T04:32:55Z
Language en_US
Faculty Griffith Health Faculty
Subject Biologically Active Molecules
URI http://hdl.handle.net/10072/47670
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1x

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