Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters

Enzyme Microb Technol. 2019 Jun:125:29-36. doi: 10.1016/j.enzmictec.2019.03.001. Epub 2019 Mar 4.


ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.

Keywords: Bioreduction; Crystal structure; Iterative saturation mutagenesis; Ketoreductase; Short-chain dehydrogenase/reductase.

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Alcohols / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites / genetics
  • Biocatalysis
  • Chryseobacterium / enzymology*
  • Chryseobacterium / genetics
  • Crystallography, X-Ray
  • Gain of Function Mutation
  • Ketones / chemistry
  • Ketones / metabolism*
  • Kinetics
  • Molecular Docking Simulation
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Protein Engineering
  • Structure-Activity Relationship


  • Alcohols
  • Bacterial Proteins
  • Ketones
  • Alcohol Oxidoreductases