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(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.
Slessor
Kate E
KE
School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Brisbane QLD 4072, Australia.
Stok
Jeanette E
JE
Cavaignac
Sonia M
SM
Hawkes
David B
DB
Ghasemi
Younes
Y
De Voss
James J
JJ
D055162:Biocatalysis; D002954:Citrobacter / Q000201:enzymology*; D003001:Cloning, Molecular; D003511:Cyclohexanols / Q000378:metabolism*; D000077591:Eucalyptol; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D039821:Monoterpenes / Q000378:metabolism*; D009243:NAD / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D003511:Cyclohexanols; C471213:His-His-His-His-His-His; D039821:Monoterpenes; D009842:Oligopeptides; D011994:Recombinant Proteins; D009243:NAD; D006639:Histidine; D010088:Oxidoreductases; D000077591:Eucalyptol