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Abstract: Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6β-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)-6β-hydroxycineole in yields of ∼1g/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. [Copyright &y& Elsevier]

Cytochrome P450cin catalyzes the monooxygenation of 1 ,8-cineole, which is structurally very similar to D-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1 ,8-cineole and D-camphor are C10 monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 Å resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cm uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe2S2 redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3. [ABSTRACT FROM AUTHOR]