Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data.

The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.

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The environmental pollutant 4-sec-butylphenol (4-sec-BP) which possesses estrogenic properties was transformed by the aerobic Gram-positive bacteria Mycobacterium neoaurum and Nocardia cyriacigeorgica into three main products (P1-P3) which were isolated and structurally characterized in detail. Two of them were products of a process resembling anaerobic metabolism of alkylphenols based on modifications of the alkyl side chain of 4-sec-BP. The first product (P1) was identified as 4-(2-hydroxy-1-methylpropyl)-phenol. The second product P2 was isolated as a mixture of at least four structures which could be identified as I 4-sec-butylidenecyclohexa-2,5-dienone; II 4-(1-methylenepropyl)-phenol; III 4-(1-methylpropenyl)-phenol; and IV 4-(1-methylallyl)-phenol. In contrast to P1 and P2, the third product (P3) resulted from a modification of the hydroxyl group of 4-sec-BP. This product was only formed by M. neoaurum and was identified as the glucoside conjugate 4-sec-butylphenol-α-D-glucopyranoside. Since in general, fungi synthesize sugar conjugates to detoxify hazardous pollutants, the formation of this conjugate is a peculiarity of M. neoaurum. Thus, altogether, six products were formed from 4-sec-BP and different transformation pathways are introduced. The hydroxylating and glucosylating capacity of the characterized bacteria open up applications in environmental protection.