Substrate and Inhibitor Spectra of Ethylbenzene Dehydrogenase: Perspectives on Application Potential and Catalytic Mechanism

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (<jats:italic>S</jats:italic>)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.</jats:p>

Anaerobic Activation of p -Cymene in Denitrifying Betaproteobacteria: Methyl Group Hydroxylation versus Addition to Fumarate

“…The reaction mechanism of ethylbenzene dehydrogenase from "A. aromaticum" EbN1 has already been intensively studied based on its crystal structure (56) and ab initio and quantum chemical modeling (65,66), as well as by substrate and inhibitor studies (67). Essentially, methylene group hydroxylation proceeds via two one-electron transfer The only other known enzyme catalyzing an O 2 -independent hydroxylation is the long-studied p-cresol methylhydroxylase (Pch) from anaerobically grown P. putida NCIB 9866, targeting the benzylic methyl group of the phenolic compound p-cresol (24,25).…”

“…Based on the above-described mechanism for ethylbenzene dehydrogenase via a "carbocation-type transition state" (56,(65)(66)(67), it is useful to consider heterolytic COH bond dissociation energies in the context of the hydroxylation of p-cymene. Values for formation of a carbenium ion and a hydride have been reported to decrease in the following order: toluene (992 kJ mol Ϫ1 [72] [73]).…”

Anaerobic Degradation of <b><i>p</i></b>-Alkylated Benzoates and Toluenes

“…The archetype of these intriguing enzymes is ethylbenzene dehydrogenase of 'A. aromaticum' EbN1, originally discovered by enzymatic and proteogenomic studies [Kniemeyer and Heider, 2001;Rabus et al, 2002] and subsequently investigated in detail by structural analysis [Kloer et al, 2006] and various modeling approaches [Szaleniec et al, 2010, as well as by substrate and inhibitor studies [Knack et al, 2012]. From these investigations a reaction mechanism for ethylbenzene dehydrogenase was inferred.…”

Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation

“…toluene). Further mixedtype inhibitors included anisole, which contains an O atom at the site to be hydroxylated, the heterocyclic analog 4-ethylpyridine or the o -thiol substituted analog of ethylbenzene, which caused a highly efficient inhibition at low concentrations [Knack et al, 2012]. Especially interesting inhibitors of EBDH were the azaborine analogs N-and B-ethyl-1,2-azaborine, where two of the ring carbon atoms are replaced by a B and an N atom.…”

“…Substrates and Industrial Applicability Although the catalytic efficiency of EBDH towards the natural substrate ethylbenzene is 10-to 100-fold larger than for any other substrate (due to the low Km value), more than 30 further substrate analogs are currently known to be turned over with very high activities [Knack et al, 2012;Szaleniec et al, 2007]. These include other hydrocarbons like propylbenzene and its unsaturated derivatives 3-phenylprop-1-ene and 3-phenylprop-1-yne or the bicyclic compounds indane, tetrahydronaphthalene or 2-ethylnaphthalene, as well as ring-substituted analogs of ethyl-or propylbenzene with hydroxyl-, amino-, fluoro-, methyl-, ethyl-or ethoxy-substituents and heterocyclic analogs like ethylfuran or ethylthiophene [Szaleniec et al, 2007.…”