Cloning and characterization of a p-cymene monooxygenase from Pseudomonas chlororaphis subsp. aureofaciens.

p-Cymene monooxygenase is the enzyme system that catalyzes the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol (p-cumic alcohol), the initial step in the assimilation of p-cymene by Pseudomonas chlororaphis subsp. aureofaciens. Cloning and sequencing of single NADH-dependent cytochrome c reductase gene (cymA) present in P. chlororaphis subsp. aureofaciens was described earlier. In this study, analysis of the upstream sequence of cymA revealed two open reading frames, designated as cymB (495 bp) and cymM (1128 bp). Database searches with the cymM gene product showed similarity to integral-membrane di-iron enzymes, while that with cymB showed no significant similarity to other known proteins with the exception of epoxystyrene isomerases. Expression of all three components (cymMBA) in Escherichia coli confirmed its ability for p-cymene methyl group hydroxylation, while expression of cymM and cymA along with the partially truncated cymB gene showed an 85% decrease in the hydroxylation capacity. Our results suggest for the first time that the small protein, CymB, having no conserved domains in protein databases, is involved as enhancer/activator in p-cymene hydroxylation.