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 investigation...