D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme

J Mol Biol. 2006 Jan 27;355(4):722-33. doi: 10.1016/j.jmb.2005.10.072. Epub 2005 Nov 14.


The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cacodylic Acid / chemistry
  • Cloning, Molecular
  • Conserved Sequence
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry
  • Gene Expression / genetics
  • Humans
  • Hydroxybutyrate Dehydrogenase / antagonists & inhibitors
  • Hydroxybutyrate Dehydrogenase / chemistry*
  • Hydroxybutyrate Dehydrogenase / genetics
  • Hydroxybutyrate Dehydrogenase / metabolism*
  • Ligands
  • Molecular Sequence Data
  • NAD / chemistry
  • NAD / metabolism
  • Protein Structure, Quaternary
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Pseudomonas fragi / enzymology*
  • Pseudomonas fragi / genetics
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • Enzyme Inhibitors
  • Ligands
  • Protein Subunits
  • NAD
  • Cacodylic Acid
  • Hydroxybutyrate Dehydrogenase

Associated data

  • PDB/1WMB
  • PDB/1X1T