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

Autor: Slessor KE; School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Brisbane QLD 4072, Australia., Stok JE, Cavaignac SM, Hawkes DB, Ghasemi Y, De Voss JJ
Jazyk: angličtina
Zdroj: Bioorganic chemistry [Bioorg Chem] 2010 Apr; Vol. 38 (2), pp. 81-6. Date of Electronic Publication: 2009 Dec 16.
DOI: 10.1016/j.bioorg.2009.12.003
Abstrakt: 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 (1R)-6beta-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)-6beta-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6beta-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6beta-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)-6beta-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.
(Copyright 2009 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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