Funktion und Mechanismus der Ethylbenzol Dehydrogenase

Typical initial reactions of anaerobic hydrocarbon degradation pathways are the hydroxylation of ethylbenzene and the addition of fumarate to toluene and to alkanes. The hydroxylation of ethylbenzene to (S)-phenylethanol initiates the anaerobic degradation of ethylbenzene in denitrifying bacteria like Aromatoleum aromaticum. This oxygen-independent and stereospecific reaction is catalyzed by ethylbenzene dehydrogenase (EbDH), a molybdenum/iron-sulfur/heme enzyme of the DMSO reductase family. In contrast, the anaerobic degradation of toluene in denitrifying bacteria like Thauera aromatica and the anaerobic degradation of alkanes in most of the sulphate-reducing bacteria is initiated by the addition of fumarate to the methyl-group of toluene and to the C2 atom of alkanes, respectively. The aim of this work was the further elucidation of the reaction mechanism of ethylbenzene dehydrogenase and investigations on the enzymes application potential. Furthermore, first investigations on the identification of a novel anaerobic alkane degradation pathway in the sulphate-reducing bacterium Desulfococcus oleovorans were performed. Based on kinetic studies of substrates and computational chemistry approaches, a reaction mechanism for EbDH was proposed. In the present work further enzyme kinetic studies with alternative substrates and inhibitors were performed which support the proposed reaction mechanism. The specific characteristics and kinetics of the substrates and inhibitors, respectively, and the nature of the hydroxylation products of the substrates, provide strong indication for the existence of the proposed radical- and carbocation-intermediate species in the catalytic mechanism of the enzyme. The results show that nature and position of specific substituents on the benzylic ring of substrates influences the reactivity with EbDH in a positive or negative way by stabilizing or destabilizing the proposed radical and/or carbocation intermediates. In contrast, the energy barriers for the formation of the radical- and/or the carbocation- intermediates cannot be overcome with EbDH inhibitors. In addition, in this work a novel class of inhibitors, which are BN-CC isosteres of ethylbenzene, was identified (Azaborines). The investigations show for the first time that artificial azaborine derivatives of organic compounds have a biochemical reactivity and provide the proof of concept that BN-CC isosterism can lead to novel compounds with novel properties. A further study of this work was the determination of the enantioselectivity of the hydrogen abstraction from the C1 atom of the ethyl-substituent of ethylbenzene as the initial step of the reaction mechanism. In doing so, specific deuterated ethylbenzene derivatives were synthesized which carry a deuterium atom instead of a hydrogen atom on the C1 atom, either in (S) or (R) configuration. With kinetic investigations, specific kinetic isotope effects were determined which indicate that the pro(S) hydrogen atom is abstracted by the enzyme in the initial step of the reaction. For the determination of the kinetic model of the EbDH reaction mechanism, enzyme kinetic investigations with two specific substrates and two specific artificial electron acceptors were performed. The evaluation of the data revealed that the enzyme follows most likely a Ping Pong mechanism. The sulphate-reducing bacterium Desulfococcus. oleovorans uses not a fumarate addition reaction as initial step of anaerobic alkane degradation, as other alkane-degrading microorganism do, but a so far unknown reaction. For the identification of this reaction, D. oleovorans was grown anaerobically on alkanes and fatty acids as carbon and electron donor. With cell free extract of these cultures, proteomic analyses were performed. It was observed that three proteins were exclusively induced in the cultures grown on alkanes. The analysis of the induced proteins by mass spectrometry showed that the proteins are the subunits of an ethylbenzene dehydrogenase-like enzyme. The amino acid sequence of the enzyme shows a high sequence identity to ethylbenzene dehydrogenase. For this reason, it is proposed that anaerobic alkane degradation in D. oleovorans is initiated by an oxygen-independent hydroxylation of alkanes.