Analysis of Essential Isoprene Metabolic Pathway Proteins in Variovorax sp. Strain WS11.

Isoprene monooxygenase (IsoMO, encoded by ) initiates the oxidation of the climate-active gas isoprene, with the genes and nearly always found adjacent to in extant and metagenome-derived isoprene degraders. The roles of and are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, sp. strain AD45 and sp. strain WS11. The genes and from and from were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione -transferases from , IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from and were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in , is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes . In isoprene-degrading bacteria, a second gene cluster, , is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera and