3-Hydroxyisobutyrate dehydrogenase from Pseudomonas putida E23: purification and characterization

Biosci Biotechnol Biochem. 1996 Dec;60(12):2043-7. doi: 10.1271/bbb.60.2043.


The NAD(+)-dependent 3-hydroxyisobutyrate dehydrogenase [EC] was purified to homogeneity from Pseudomonas putida E23. The enzyme was a tetramer (molecular mass, 120 kDa) consisted of identical subunits (molecular mass, 30 kDa). The enzyme was specific for NAD+ (Km, 0.44 mM). The maximal activity was obtained at about pH 10. The enzyme was specific for the L-isomer of 3-hydroxyisobutyrate. In addition to L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were substrates. The Km for L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were 0.12, 18, 44, and 83 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and AgNO3, but not by EDTA, alpha,alpha'-dipyridyl, and o-phenanthroline. The N-terminal 26 amino acid sequence was compared with the sequences deduced from the enzyme genes of rat liver and Pseudomonas aeruginosa.

MeSH terms

  • Alcohol Oxidoreductases / analysis*
  • Alcohol Oxidoreductases / antagonists & inhibitors
  • Alcohol Oxidoreductases / biosynthesis
  • Amino Acid Sequence
  • Animals
  • Coenzymes / metabolism
  • Culture Media
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Pseudomonas putida / enzymology*
  • Rats
  • Soil Microbiology
  • Spectrophotometry, Ultraviolet
  • Substrate Specificity


  • Coenzymes
  • Culture Media
  • Alcohol Oxidoreductases
  • 3-hydroxyisobutyrate dehydrogenase