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

FEBS J. 2022 Feb;289(4):1023-1042. doi: 10.1111/febs.16216. Epub 2021 Oct 14.


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 BbsC2 and BbsD2 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)-2-benzoylsuccinyl-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.

Keywords: beta oxidation; pseudoenzyme; short-chain dehydrogenases/reductases; structural biology; toluene metabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / biosynthesis
  • Acyl Coenzyme A / chemistry
  • Biocatalysis
  • Models, Molecular
  • Molecular Structure
  • Short Chain Dehydrogenase-Reductases / metabolism*
  • Succinates / chemistry
  • Succinates / metabolism
  • Sulfhydryl Compounds / chemistry
  • Sulfhydryl Compounds / metabolism
  • Thauera / enzymology*
  • Toluene / chemistry
  • Toluene / metabolism*


  • Acyl Coenzyme A
  • Succinates
  • Sulfhydryl Compounds
  • benzylsuccinate
  • Toluene
  • Short Chain Dehydrogenase-Reductases