Characterization of the Gene Cluster Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

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Abstract
The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S -transferase with activity towards 1,2-epoxy-2-methyl-3-butene ( isoI ) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase ( isoH ). Furthermore, a gene encoding a second glutathione S -transferase was identified ( isoJ ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1,2-epoxy-2-methyl-3-butene. Downstream of isoJ , six genes ( isoABCDEF ) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene ( isoG ) showed significant similarity with that of α-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.