| Due to the relatively high water solubility of the gasoline components known as BTEX (benzene, toluene, ethylbenzene, and the xylenes), these compounds readily infiltrate aquifers underlying gasoline spills and contaminate groundwaters. Intrinsic bioremediation is one means by which to remediate the contamination. Since contaminated aquifers are often rendered anaerobic by the expeditious actions of aerobic microorganisms, anaerobic microorganisms must be relied upon to restore contaminated sites. The objective of this research was to provide a fundamental understanding of anaerobic ethylbenzene degradation.; Initial reactions in the anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, Azoarcus sp. strain EB1. Based upon growth and cell suspension experiments, a pathway for anaerobic ethylbenzene oxidation was proposed in which ethylbenzene oxidation is initiated by dehydrogenation of ethylbenzene at the methylene carbon to form (S)-(−)-1-phenylethanol. (S)-(−)-1-Phenylethanol is further oxidized to acetophenone. Acetophenone may then be carboxylated to form benzoyl acetate. Thiolytic cleavage of activated benzoyl acetate (benzoyl acetyl-CoA) would produce benzoyl-CoA, a common intermediate of anaerobic aromatic metabolism, and acetyl-CoA, a central metabolite.; We examined the in vitro enzymatic activities of ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase, the enzymes catalyzing the first two reactions in anaerobic ethylbenzene oxidation. The inability of cell-free extracts to transform styrene in combination with experiments using ethylbenzene-djo as substrate suggest that styrene is not a free intermediate in the oxidation of ethylbenzene to 1-phenylethanol. Ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate grown cells.; We purified the membrane-associated ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (α β γ) with subunits of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol molybdenum, 16 mol iron and 15 mol acid-labile sulfur per mol holoenzyme, as well as a molybdopterin cofactor. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the DMSO reductase family of molybdopterin-containing enzymes. |
