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

Abstract

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 cin has previously been demonstrated to perform the first oxidation to produce (1 R)-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 (1 R)-6β-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1 R)-6β-hydroxycineole was converted to (1 R)-6-ketocineole. The (1 R)-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 (1 R)-6-ketocineole to (1 R)-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 2+ ions or proteolytic removal of the His-tag.