Abstract The first intermediate of anaerobic toluene ca- tabolism, (R)-benzylsuccinate, is formed by enzymic ad- dition of the methyl group of toluene to a fumarate cosub- strate and is subsequently activated to (R)-2-benzylsuc- cinyl-CoA. This compound is then oxidised to benzoyl- CoA and succinyl-CoA by a specific β-oxidation path- way. The enzyme catalysing the first oxidation step of this pathway, (R)-benzylsuccinyl-CoA dehydrogenase, is en- coded by the bbsG gene in Thauera aromatica. It was func- tionally overproduced in Escherichia coli, purified and characterised. The enzyme is a homotetramer with a sub- unit size of 45 kDa and contains one FAD per subunit. It is highly specific for (R)-benzylsuccinyl-CoA and is in- hibited by (S)-benzylsuccinyl-CoA. An apparent K m value of 110±10 μM was obtained for (R)-benzylsuccinyl-CoA. The reaction product of (R)-benzylsuccinyl-CoA dehydro- genase was identified as (E)-benzylidene-succinyl-CoA by comparison with the chemically synthesised compound, which was obtained via a new synthetic procedure. (R)-Ben- zylsuccinyl-CoA dehydrogenase was detected as a specifi- cally substrate-induced protein in toluene- and m-xylene- grown cells of several bacterial species, using enzyme ac- tivity and immunological detection. Keywords Anaerobic toluene metabolism · Benzylsuccinyl-CoA · Benzylidene-succinyl-CoA · Phenylitaconyl-CoA · acyl-CoA dehydrogenase · Flavin · Stereochemistry · β-Oxidation Abbreviations BCDH Benzylsuccinyl-CoA dehydrogenase · ETF Electron-transferring flavoprotein · UQ Ubiquinone · BS Benzylsuccinate · B en S Benzylidene succinate/phenylitaconate · Fc + Ferricenium Introduction It has been known for only about a decade that some bac- teria degrade aromatic hydrocarbons, such as toluene, in the absence of oxygen. The metabolic pathway of anaero- bic toluene catabolism has recently been elucidated (for a review, see Heider et al. 1999). Overall, toluene is oxi- dised to benzoyl-CoA, a common intermediate in anaero- bic catabolism of many aromatic compounds (reviewed in Heider and Fuchs 1997). Anaerobic toluene catabolism is initiated by formation of (R)-benzylsuccinate as first in- termediate via an unusual addition reaction of the methyl group of toluene to the double bond of a fumarate cosub- strate (Biegert et al. 1996; Beller and Spormann 1997; Fig. 1). This reaction is catalysed by (R)-benzylsuccinate synthase, a novel glycyl-radical enzyme (Coschigano et al. 1998; Leuthner et al. 1998; Beller and Spormann 1999). This type of reaction seems to be highly significant in anaerobic bacterial metabolism, since analogous fumarate addition reactions were recognised as initial reactions in the anaerobic catabolic pathways of m-xylene (Krieger at al. 1999), 2-methylnaphthalene (Annweiler et al. 2001), m- and p-cresol (Müller et al. 1999, 2001), and even of n-alkanes (Rabus et al. 2001). Further anaerobic toluene metabolism involves a spe- cific β-oxidation pathway for (R)-benzylsuccinate, which yields benzoyl-CoA and regenerates succinyl-CoA. All en- zymes of this β-oxidation pathway are apparently encoded in a common operon of nine genes (Leuthner and Heider 2000). (R)-Benzylsuccinate is first activated to (R)-2-ben- zylsuccinyl-CoA by a novel succinyl-CoA-dependent CoA-transferase (Leutwein and Heider 1999, 2001, Fig. 1). The activated CoA-thioester is then oxidised to benzoyl- CoA and succinyl-CoA in four successive reactions. The first oxidation step of (R)-2-benzylsuccinyl-CoA was proposed to yield (E)-benzylidene-succinyl-CoA [trivial name: (E)-phenylitaconyl-CoA], based on accumulation of (E)-benzylidene-succinate in supernatants of growing cultures and permeabilised cells (Chee-Sanford et al. 1996; Beller and Spormann 1997; Krieger et al. 1999). The gene Christina Leutwein · Johann Heider (R )-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway Received: 12 June 2002 / Revised: 29 August 2002 / Accepted: 30 August 2002 / Published online: 8 October 2002 ORIGINAL PAPER C. Leutwein · J. Heider (✉) Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität, Schänzlestrasse 1 79104 Freiburg, Germany e-mail: heiderj@uni-freiburg.de, Tel.: +49-761-2032774, Fax: +49-761-2032626 Arch Microbiol (2002) 178 : 517–524 DOI 10.1007/s00203-002-0484-5 © Springer-Verlag 2002