Structural basis for substrate specificity of methylsuccinyl-CoA dehydrogenase, an unusual member of the acyl-CoA dehydrogenase family

J Biol Chem. 2018 Feb 2;293(5):1702-1712. doi: 10.1074/jbc.RA117.000764. Epub 2017 Dec 22.


(2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD) and is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation. It catalyzes the oxidation of (2S)-methylsuccinyl-CoA to α,β-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. Here we report the crystal structure of MCD at a resolution of 1.37 Å. The enzyme forms a homodimer of two 60-kDa subunits. Compared with other ACDs, MCD contains an ∼170-residue-long N-terminal extension that structurally mimics a dimer-dimer interface of these enzymes that are canonically organized as tetramers. MCD catalyzes the unprecedented oxidation of an α-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. MCD apparently evolved toward preventing the nonspecific oxidation of succinyl-CoA, which is a close structural homolog of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism. For different metabolic engineering and biotechnological applications, however, an enzyme that can oxidize succinyl-CoA to fumaryl-CoA is sought after. Based on the MCD structure, we were able to shift substrate specificity of MCD toward succinyl-CoA through active-site mutagenesis.

Keywords: X-ray crystallography; acyl-CoA dehydrogenase; bioengineering; enzyme catalysis; enzyme kinetics; enzyme mutation; enzyme purification; enzyme structure; ethylmalonyl-CoA pathway; flavin adenine dinucleotide (FAD); glutaryl-CoA dehydrogenase; isobutyryl-CoA dehydrogenase; mesaconyl-CoA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Oxidation-Reduction
  • Oxidoreductases / chemistry*
  • Paracoccus denitrificans / enzymology*
  • Protein Domains
  • Protein Structure, Quaternary
  • Structure-Activity Relationship
  • Substrate Specificity


  • Bacterial Proteins
  • Oxidoreductases

Associated data

  • PDB/1JQI
  • PDB/3MDE
  • PDB/3B96
  • PDB/1IVH
  • PDB/1RX0
  • PDB/3MPI
  • PDB/6ES9
  • PDB/4N5F