Anaerobic Toluene Catabolism of Thauera aromatica : the bbs Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate

Abstract

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent ␤-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in ␤ oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the ␤-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

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

“…Further steps in benzylsuccinate degradation detected by enzymatic assays are a CoA-transferase reaction yielding benzylsuccinyl-CoA and a subsequent dehydrogenase reaction leading to phenylitaconyl-CoA (Leutwein and Heider 1999. The subsequent degradation steps have been proposed on the basis of molecular investigations of the bbs operon (Leuthner and Heider 2000) and it was hypothesize that succinyl-CoA is cleaved from benzoylsuccinyl-CoA to be recycled for further addition reactions and results in the formation of the central intermediate benzoyl-CoA. In contrast, recent investigations on anaerobic degradation of n-hexan proposed a decarboxylation reaction prior to stepwise β-oxidation (Widdel and Rabus 2001;Wilkes et al 2002).…”

Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2

“…W. Murdoch and A. G. Hay Although there are over 2000 SCPx-type bacterial genes in the NCBI database, only four have been characterized to any degree: ditF (Pseudomonas abietaniphila), phlC (P. putida) (Bangera & Thomashow, 1999), ORF-2 from the camphor catabolic cluster (Rhodococcus sp. NCIMB 9784) (Grogan et al, 2001;Roberts et al, 2004), and bbsB from both Aromatoleum aromaticum EbN1 (Kube et al, 2004;Kühner et al, 2005) and Thauera aromatica (Leuthner & Heider, 2000). As part of a pathway for the metabolism of toluene to benzoyl-CoA under anaerobic denitrifying conditions, BbsB in conjunction with BbsA performs the decondensation of an a-aliphatic b-keto CoA adduct in a manner that mirrors the role of eukaryotic SCPx in the metabolism of bile acids and branched fatty acids (Fig.…”

“…2) we hypothesize that IpfD and E are responsible for the acid side chain removal based on the role that related proteins play in acyl group transfer (Fig. 4) (Bangera & Thomashow, 1999;Kube et al, 2004;Kühner et al, 2005;Leuthner & Heider, 2000;Verhoeven & Jakobs, 2001;Wanders et al, 1997;Westin et al, 2007).…”

Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

“…1A) (5). The addition of fumarate serves to activate the benzylic carbon of toluene, allowing it to be metabolized through ␤ oxidation to benzoyl-CoA and then reductively dearomatized by benzoylCoA reductase before further oxidation (4,6).…”