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Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

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 alpha-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.

SUBMITTER: van Hylckama Vlieg JE

PROVIDER: S-EPMC101893 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

van Hylckama Vlieg J E JE Leemhuis H H Spelberg J H JH Janssen D B DB

Journal of bacteriology 20000401 7

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 gluta ...[more]

PMID: 10715003

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Project description:Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.

Project description:Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus COMAMONAS: Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3' end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Km(r) cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of sigma(54)-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-beta-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

Dataset Information

Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

Publications

Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

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