Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T

Abstract

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.Interest in the anaerobic metabolism of alkylbenzenes is increasing because of the subject's relevance to bioremediation of fuel-contaminated aquifers and because of novel aspects of the biochemical reactions involved. Of particular biochemical interest is how aromatic hydrocarbons can be activated in the absence of mono-and dioxygenase-catalyzed reactions. Toluene appears to be the most readily degraded of the alkylbenzenes under anaerobic conditions and has been the most intensively studied. Since 1989, pure cultures capable of anaerobic toluene degradation have been reported under denitrifying conditions (10,13,15,21,22), ferric iron-reducing conditions (17, 18), and sulfate-reducing conditions (5, 20); fermentative-methanogenic mixed enrichment cultures that degrade toluene have also been reported (11,16,24). Several anaerobic toluene mineralization pathways have been proposed, with varying degrees of experimental support. Initial reactions of toluene in these proposed pathways include (i) hydroxylation of the methyl group to form benzyl alcohol (1, 2), (ii) hydroxylation of the aromatic ring to form p-cresol (e.g., references 16 and 24), (iii) attack of the methyl carbon by acetyl coenzyme A (CoA) to form phenylpropionyl-CoA (12), and very recently (iv) addition of the methyl carbon to fumarate to form benzylsuccinate (7). To date,...

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

“…The specific activity of the cell extract at 4 °C was 2.4 nmol/min/mg of protein, whereas at 30 °C the specific activity rose to 7.4 nmol/min/mg. This level of activity is similar to that reported in permeablized cells of Azoracus sp (18), and some 4 orders of magnitude higher than that reported for purified enzyme from T. aromatica (6). As judged by SDS-PAGE, the BSS α-subunit, a conspicuous band of M r 98,000, comprised about 3.5 % of the total proteins present in the cell free extract, which allows us to estimate k cat for BSS as approximately 0.11 s −1 at 4 °C and 0.35 s −1 at 30°C .…”

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

“…HxN1, which actives n-alkanes by using Ass (Mas), can co-activate cyclo-alkanes in the presence of n-alkanes (Wilkes et al, 2003). Similarly, other Ass enzymes can activate toluene in the presence of n-alkanes (Rabus et al, 2011), whereas Bss can catalyze fumarate addition to both toluene and xylenes (Beller and Spormann, 1997).…”

Degradation of o -xylene and m -xylene by a novel sulfate-reducer belonging to the genus Desulfotomaculum

“…Later, transformation of o-xylene to o-methyl homologs of benzylsuccinate by toluene-degrading strains was reported; however, no further oxidation steps could be observed (Beller and Spormann 1997a). So far, only one anaerobic o-xylene-degrading bacterium, the sulfate-reducing strain oXyS1, has been isolated from marine sludge (Harms et al 1999 …”