Rational design of Meso-2,3-butanediol dehydrogenase by molecular dynamics simulation and experimental evaluations

FEBS Lett. 2017 Oct;591(20):3402-3413. doi: 10.1002/1873-3468.12834. Epub 2017 Sep 17.

Abstract

Meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) catalyzes NAD+ -dependent conversion of meso-2,3-butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso-2,3-BDH was identified. The two short α helixes positioned away from the α4-helix possibly expose the hydrophobic ligand-binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA-binding free energy analysis shows that Phe212 and Asn146 function as the key product-release sites. Site-directed mutagenesis experiments targeted to the sites show that the kcat of Phe212Tyr is enhanced up to (4-8)-fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short-chain dehydrogenases/reductases.

Keywords: meso-2,3-butanediol dehydrogenase; rational design; short-chain dehydrogenase/reductase.

Publication types

  • Letter

MeSH terms

  • Acetoin / chemistry
  • Acetoin / metabolism
  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Butylene Glycols / chemistry
  • Butylene Glycols / metabolism
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression
  • Kinetics
  • Klebsiella pneumoniae / enzymology
  • Klebsiella pneumoniae / genetics*
  • Models, Molecular
  • Molecular Docking Simulation*
  • Molecular Dynamics Simulation*
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Engineering
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermodynamics

Substances

  • Bacterial Proteins
  • Butylene Glycols
  • Recombinant Proteins
  • 2,3-butylene glycol
  • Acetoin
  • Alcohol Oxidoreductases
  • butanediol dehydrogenase