Awakening the Sleeping Carboxylase Function of Enzymes: Engineering the Natural CO 2-Binding Potential of Reductases

J Am Chem Soc. 2019 Jun 26;141(25):9778-9782. doi: 10.1021/jacs.9b03431. Epub 2019 Jun 14.


Developing new carbon dioxide (CO2) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology. Here we used bioinformatics to identify a "sleeping carboxylase function" in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. We show that propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO2 and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO2-binding and kinetic parameters comparable to naturally existing CO2-fixing enzymes. Our results provide a strategy to develop novel CO2-fixing enzymes and shed light on the emergence of natural carboxylases during evolution.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaea / enzymology
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Carbon Cycle
  • Carbon Dioxide / chemistry
  • Carbon Dioxide / metabolism
  • Carboxy-Lyases / chemistry*
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism
  • Catalytic Domain / genetics
  • Kinetics
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism
  • Sphingomonadaceae / enzymology
  • Water / chemistry
  • Water / metabolism


  • Archaeal Proteins
  • Bacterial Proteins
  • Water
  • Carbon Dioxide
  • Oxidoreductases
  • acryloyl-CoA reductase
  • Carboxy-Lyases