Inactive pseudoenzyme subunits in heterotetrameric BbsCD, a novel short‐chain alcohol dehydrogenase involved in anaerobic toluene degradation

Abstract

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as first intermediate, which is further degraded via β-oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)-benzylidenesuccinyl-CoA hydratase (BbsH), and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl-CoA hydratase family, converts (E)-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short-chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2-symmetric heterotetramer consisting of BbsC 2 and BbsD 2 dimers. BbsD subunits are catalytically active and capable of binding NAD + and substrate, whereas BbsC subunits represent built-in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)-diastereomer of 2-(α-hydroxybenzyl)succinyl-CoA to (S)-2benzoylsuccinyl-CoA by hydride transfer to the re-face of nicotinamide adenine dinucleotide (NAD) + . Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family, which adopt a heterotetrameric architecture and catalyze the β-oxidation of aromatic succinate adducts.

Finis tolueni: a new type of thiolase with an integrated Zn‐finger subunit catalyzes the final step of anaerobic toluene metabolism

“…The optimum pH for benzoylsuccinyl-CoA formation from benzoyl-CoA and succinyl-CoA was at pH 6.4, and HPLC analyses confirmed that BbsAB catalyzed the time-dependent decrease of succinyl-CoA and benzoyl-CoA with a simultaneous increase of a new compound, which comigrated with benzoylsuccinyl-CoA obtained in another study (Fig. 2C) [10]. The chemical nature of this product as a ketone-containing compound was further supported by converting it to a hydrazone by adding phenylhydrazine, resulting in a shift of the retention time (Fig.…”

“…BbsAB is expected to turn over ( S )‐2‐benzoylsuccinyl‐CoA as substrate, because this diastereomer was found to be produced by BbsCD, the enzyme preceding BbsAB in the pathway (Fig. 1) [10]. Therefore, ( S )‐2‐benzoylsuccinyl‐CoA was modelled based on the geometric/electrostatic properties of the active site of BbsAB thereby starting from the firmly bound CoA.…”

“…Activity of the proteins as benzoylsuccinyl-CoA thiolase was determined by measuring the reverse reaction via high performance liquid chromatography (HPLC) analysis, namely the condensation of benzoyl-CoA and succinyl-CoA to benzoylsuccinyl-CoA, because benzoylsuccinyl-CoA is neither commercially available nor easily accessible via chemical synthesis. No activity was recorded in recombinant cell extracts or DEAE fractions because thioesterase activity leads to rapid hydrolysis of benzoylsuccinyl-CoA [10]. Likewise.…”

“…It is first activated to 2-(R)-benzylsuccinyl-CoA by the CoA-transferase BbsEF [7,8], then oxidized to (E)-benzylidenesuccinyl-CoA by the acyl-CoA dehydrogenase BbsG [9]. Subsequently, water is added at the double bond by enoyl-CoA hydratase BbsH to generate the b-hydroxyacyl compound (S, R)-2-(a-hydroxybenzyl)succinyl-CoA, which is further oxidized to (S)-2-benzoylsuccinyl-CoA by the shortchain dehydrogenase BbsCD [10]. The final step of the pathway is a thiolytic cleavage of (S)-2-benzoylsuccinyl-CoA to succinyl-CoA and benzoyl-CoA [6] (Fig.…”

“…The biochemistry of the first four enzymes of the benzylsuccinate degradation pathway has already been investigated [8][9][10]. To complete the understanding of the pathway and the enzymatic reactions involved, we focus in this study on the biochemical and structural properties of the last enzyme of the pathway, benzoylsuccinyl-CoA thiolase (EC 2.3.1.-).…”