Significance of protein-substrate hydrogen bonding for the selectivity of P450-catalysed oxidations

P450 and P450 are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein–substrate H bonding has been previously proposed as crucial in the selectivity of P450 oxidations, but not as essential for P450 . To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein–substrate H bond to selectivity in both P450 and P450 . The H bonding in P450 was re-examined using camphane, another substrate for which the protein–substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein–substrate H bond was not a factor in substrate affinity for the enzyme

Cloning, expression and purification of cindoxin, an unusual FMN-containing cytochrome P450 redox partner

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450 (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range

Electrochemistry of P450cin: new insights into P450 electron transfer

Electrochemistry of bacterial cytochrome P450cin (CYP176A) reveals that, unusually, substrate binding does not affect the heme redox potential, although a marked pH dependence is consistent with a coupled single electron/single proton transfer reaction in the range 6 < pH < 10

Cineole biodegradation: Molecular cloning, expression and characterisation of (1R)-6beta - hydroxycineole dehydrogenase from Citrobacter braakii

The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450 has previously been demonstrated to perform the first oxidation to produce (1R)-6β-hydroxycineole. In this study, we have cloned cinD from C. braakii and expressed the gene product, which displays significant homology to a number of short-chain alcohol dehydrogenases. It was demonstrated that the gene product of cinD exhibits (1R)-6β-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6β-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6β-hydroxycineole dehydrogenase exhibited a strict requirement for NAD(H), with no reaction observed in the presence of NADP(H). The enzyme also catalyses the reverse reaction, reducing (1R)-6-ketocineole to (1R)-6β-hydroxycineole. During this study the N-terminal His-tag used to assist protein purification was found to interfere with NAD(H) binding and lower enzyme activity. This could be recovered by the addition of Ni ions or proteolytic removal of the His-tag

An in vivo cytochrome P450(cin) (CYP176A1) catalytic system for metabolite production

Cytochrome P450 (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6β-hydroxycineole. P450 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 , cindoxin and E. coli flavodoxin reductase and a bicistronic plasmid that encodes only P450 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

Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s

P450cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450cin D241N mutant has been produced and found to be analogous to the P450cam D251N mutant. P450 cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase