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

Abstract

Cytochrome P450 cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6β-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)-6β-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.

Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450‐Catalysed Oxidations

“…In each product, a single oxygen had been incorporated as shown by the observed molecular ion ( m / z 184) in their mass spectra. To determine the regiochemistry of oxygenation of thiocamphor by P450 cin the reported whole cell system expressing catalytically active P450 cin was used to generate sufficient quantities of the thiocamphor oxidation products for structure elucidation. The products were isolated from the in vivo culture, purified by flash chromatography and further characterised by 1 H and 13 C NMR spectroscopy.…”

“…Thus, in contrast to the oxidation of camphor 1 and camphane 7 by P450 cin , in which at least five or six different oxidised products are formed, respectively the oxidation of thiocamphor 3 by P450 cin appears to occur specifically at the sulfur atom. This result suggests that, in the absence of H bonding, it is the reactivity of the sulfur atom that is dominant in directing substrate oxidation rather than any interaction between the substrate and the active site of P450 cin .…”

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 fromSphingobium yanoikuyaestrain B2

“…CYP101A1 produces predominantly (1 S )‐3α‐hydroxy‐1,8‐cineole and/or its enantiomer (1 S )‐5α‐hydroxy‐1,8‐cineole, whilst the main product of CYP101B1‐catalyzed oxidation is (1 S )‐3α‐hydroxy‐1,8‐cineole (nomenclature of hydroxy‐1,8‐cineoles is assigned as recently proposed by Azerad) . CYP176A1 from Citrobacter braakii has been shown to regiospecifically hydroxylate 1,8‐cineole at position 6 to afford (1 R )‐6β‐hydroxy‐1,8‐cineole . A CYP176A1 N242A mutant showed altered regio‐ and stereoselectivity, producing mainly (1 S )‐6α‐hydroxy‐1,8‐cineole, and highlights the power of structure‐based protein engineering.…”

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

“…Hydroxylation of 1,8-cineole can be achieved chemically; however, these reactions often use a range of environmentally hazardous reagents and/or yield product mixtures (4)(5)(6). On the other hand, biooxygenation of 1,8-cineole using enzymes or whole cells can be performed under mild reaction conditions with the potential to yield products with high enantiopurity (6,7).…”

“…The first bacterial 1,8-cineole-hydroxylating enzyme which was isolated and characterized was isolated from C. braakii (11). The initial oxidation of 1,8-cineole by this Gram-negative bacterium is catalyzed by a P450 designated P450 cin (CYP176A1), and with the aid of suitable electron transport partners it yielded (1R)-6␤-hydroxy-1,8-cineole (7,11). More recently, other P450s, including CYP101A1, CYP101B1, and the N252A P450 cin mutant, were shown to hydroxylate 1,8-cineole and yield a range of hydroxy-1,8-cineole derivatives (14,15).…”