Review Article J Mol Microbiol Biotechnol 2016;26:29–44 Published online: March 10, 2016 DOI: 10.1159/000441656 Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes a d c a Johann Heider Maciej Szaleniec Berta M. Martins Deniz Seyhan a, b e Wolfgang Buckel Bernard T. Golding a Laboratory of Microbial Biochemistry, LOEWE Center for Synthetic Microbiology, Philipps University Marburg, b c and Max-Planck-Institut für terrestrische Mikrobiologie, Marburg , and Institut für Biologie, Strukturbiologie/ d Biochemie, Humboldt-Universität zu Berlin, Berlin , Germany; Jerzy Haber Institute of Catalysis and Surface e Chemistry, Polish Academy of Sciences, Kraków , Poland; School of Chemistry, Newcastle University, Newcastle upon Tyne , UK Key Words Anaerobic Toluene Degradation Benzylsuccinate synthase · Fumarate-adding enzyme · Glycyl radical · Toluene · Alkane · Anaerobic toluene Hydrocarbons were long believed to be resistant to degradation microbial degradation in the absence of oxygen. It was therefore surprising to find that many bacteria degrade these compounds anaerobically [Aeckersberg et al., 1991; Abstract Dolfing et al., 1990; Rabus et al., 1993; Vogel and Grbic- The pathway of anaerobic toluene degradation is initiated Galic, 1986; Zeyer et al., 1986]. After the initial reports by a remarkable radical-type enantiospecific addition of the dating from 1986, the degradation pathways employed chemically inert methyl group to the double bond of a fuma- by these bacteria remained enigmatic for a decade. How- rate cosubstrate to yield (R) -benzylsuccinate as the first in- ever, over the last 20 years, various oxygen-independent termediate, as catalyzed by the glycyl radical enzyme ben- reactions have been characterized for the activation of zylsuccinate synthase. In recent years, it has become clear diverse hydrocarbon substrates, depending on the or- that benzylsuccinate synthase is the prototype enzyme of a ganism and the respective hydrocarbon [Booker, 2009; much larger family of fumarate-adding enzymes, which play Foght, 2008; Fuchs et al., 2011; Heider, 2007; Heider and important roles in the anaerobic metabolism of further aro- Schühle, 2013; Rabus et al., 2014]. In the case of anaero- matic and even aliphatic hydrocarbons. We present an over- bic toluene degradation, a number of competing hypo- view on the biochemical properties of benzylsuccinate syn- thetical mechanisms have been proposed as potential ini- thase, as well as its recently solved structure, and present the tiating reactions [Altenschmidt and Fuchs, 1991; Evans results of an initial structure-based modeling study on the et al., 1992; Frazer et al., 1995; Rabus and Widdel, 1995; reaction mechanism. Moreover, we compare the structure of Seyfried et al., 1994; Zeyer et al., 1986], culminating in benzylsuccinate synthase with those predicted for different the discovery of a novel biochemical reaction generating clades of fumarate-adding enzymes, in particular the paralo- benzylsuccinate, a C4 adduct of toluene, by the addition gous enzymes converting p -cresol, 2-methylnaphthalene or of a fumarate cosubstrate to the methyl group of toluene n -alkanes. © 2016 S. Karger AG, Basel [Beller and Spormann, 1997a, b; Biegert et al., 1996] © 2016 S. Karger AG, Basel Johann Heider 1464–1801/16/0263–0029$39.50/0 Laboratory of Microbial Biochemistry, Philipps University of Marburg Karl-von-Frisch-Strasse 8 E-Mail [email protected] DE–35043 Marburg (Germany) www.karger.com/mmb E-Mail heider @ biologie.uni-marburg.de BssABC COO– 2 [H] bssD bbsA bssC bbsB COO– Toluene bbsC bssA COO– Fumarate operon operon COO– bbsD bss bbs bssB COO– bbsE bssE COO– (R)-Benzylsuccinate [CoA] Succinate bbsF bssF CO SCoA BbsEF CO SCoA CO SCoA bbsJ bssG bbsG BbsG bssH COO– COO– bbsH Succinyl-CoA 2 [H] Benzoyl-CoA BbsAB (R)-Benzylsuccinyl-CoA bbsI 2 [H] CO SCoA CO SCoA O CO SCoA ring HO H O – 2 COO– reduction COO BbsCD COO– BbsH Benzoylsuccinyl-CoA (E)-Benzylidene succinyl-CoA 2 [H] (į-Hydroxybenzyl)- succinyl-CoA Fig. 1. Anaerobic toluene metabolic pathway and gene organiza- ent hatching patterns. The indicated 2- (S) , 3- (R) -enantiomeric tion. The reactions of benzylsuccinate synthase (BssABC) and the structure of the 3-hydroxyacyl-CoA intermediate has been impli- enzymes of the further β-oxidation pathway are indicated by the cated by recent structural studies on BbsH and BbsCD [Essen and respective gene products. Arrows indicate the reversibility or ir- Heider, unpubl. data]. Gray: universally conserved genes in bss op- reversibility of the respective steps. The operon organization of the erons with unknown function; white: probably nonessential genes bss and bbs operons are indicated in the left and right margins, re- of unknown function. spectively. Functions of the gene products are indicated by differ- ( fig. 1 ). Benzylsuccinate has previously been observed as and Spormann, 1997a, b; Rabus and Heider, 1998; excreted metabolite in supernatants of many toluene-de- Zengler et al., 1999]. Subsequently, the reaction was grading anaerobes, but was regarded as dead-end me- shown to be stereospecific as (R) -benzylsuccinate was ex- tabolite indicative of other metabolic pathways because clusively generated [Beller and Spormann, 1998; Leut- it did not support growth of any known anaerobic tolu- wein and Heider, 1999]. The enzyme catalyzing the for- ene degrader [Beller et al., 1992; Evans et al., 1992; Fra- mation of this compound was identified as a new type of zer et al., 1995; Seyfried et al., 1994]. The role of benzyl- glycyl radical enzyme (GRE), which was called benzyl- succinate as a true intermediate in anaerobic toluene me- succinate synthase (BSS) [Beller and Spormann, 1999; tabolism and the necessity of a fumarate cosubstrate were Leuthner et al., 1998]. At the time of its discovery, BSS first demonstrated by in vitro experiments with extracts was only the third type of GRE with a known function, of the denitrifying bacterium Thauera aromatica [Bie- akin to pyruvate formate lyase (Pfl) [Knappe and Sawers, gert et al., 1996]. These findings were confirmed with tol- 1990] and anaerobic ribonucleotide reductase [Eklund uene-degrading strains affiliated to the closely related and Fontecave, 1999; Reichard, 1997]. Today, three more genera Azoarcus and Aromatoleum , sulfate-reducing types of GREs with known functions are anaerobic coen- strains affiliated to the genus Desulfobacula and the an- zyme B12 -independent diol dehydratases [O’Brien et al., oxygenic phototroph Blastochloris sulfoviridis [Beller 2004; Raynaud et al., 2003], choline trimethylamine ly- 30 J Mol Microbiol Biotechnol 2016;26:29–44 Heider/Szaleniec/Martins/Seyhan/Buckel/ DOI: 10.1159/000441656 Golding ases [Craciun and Balskus, 2012; Craciun et al., 2014] and Benzylsuccinate Synthase 4-hydroxyphenylacetate or indoleacetate decarboxylases [Selmer and Andrei, 2001; Selvaraj et al., this vol., pp. BSS was first purified and characterized from the Be- 76–91], as well as a growing subfamily of BSS-like fuma- taproteobacteria T. aromatica and Azoarcus strain T rate-adding enzymes (FAEs) involved in the anaerobic [Beller and Spormann, 1999; Leuthner et al., 1998]. The degradation of various other chemically inert substrates enzyme catalyzes the formation of (R) -benzylsuccinate, (see below). with toluene and fumarate as the only substrates, under The downstream pathway of anaerobic toluene me- strictly anoxic conditions. The enzyme is highly specific tabolism was also identified in T. aromatica and consists for fumarate, which can only be partially replaced by ma- of a modified β-oxidation route ( fig. 1 ), which has been leate [Beller and Spormann, 1997b; Biegert et al., 1996; Li investigated by biochemical and molecular biological and Marsh, 2006a, b], but it is still an open question methods. All enzymes of this pathway are encoded by whether maleate actually enters the active site or acts in- the common, toluene-induced bbs operon (for β- directly, for example by inhibiting fumarase in the ex- oxidation of benzylsuccinate) [Leuthner and Heider, tracts (or even in the partially purified enzymes), thereby 2000], which has been identified in all studied anaerobic increasing the background fumarate concentration [Bie- toluene degraders ( fig. 1 ). Further degradation of (R) - gert et al., 1996]. Conversely, all analyzed enzymes ac- benzylsuccinate starts with its regio- and enantiospecif- cepted a number of substituted toluene derivatives, such ic activation to the CoA thioester 2- (R) -benzylsuccinyl- as fluorotoluenes [Biegert et al., 1996], o -xylene [Beller CoA by a specific CoA transferase consisting of two sub- and Spormann, 1997b], all three cresol isomers [Verfürth units encoded by the bbsEF genes [Leuthner and Heider, et al., 2004] and o -toluidine [Lippert and Heider, unpubl. 2000; Leutwein and Heider, 1999, 2001]. The β-oxidation data]. Interestingly, however, BSS orthologs from differ- of benzylsuccinyl-CoA then continues by oxidation to ent strains vary in their activities for the xylene isomers: (E) -benzylidenesuccinyl-CoA, mediated by benzyl- BSS of T. aromatica strain K172, which degrades toluene succinyl-CoA dehydrogenase (BbsG) [Leutwein and but not xylenes, does not convert any xylene isomer to the Heider, 2002]. This is followed by hydration to the cor- corresponding succinate adduct, whereas BSS of Azoar- responding 3-hydroxyacyl-CoA derivative by the enoyl- cus strain T, a known degrader of toluene or m -xylene, CoA hydratase BbsH, oxidation of the alcohol