Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum.
An NADP(+)-dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40-43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mumol min-1 mg-1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (Km) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The Km values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.
Bayer
M
M
Institute for Organic Chemistry, Technical University, Munich, Garching, Germany.
Günther
H
H
Simon
H
H
D020155:3-Hydroxybutyric Acid; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001709:Biotechnology; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
D006885:Hydroxybutyrates; D009249:NADP; D000429:Alcohol Oxidoreductases; C092151:(S)-3-hydroxycarboxylate oxidoreductase; D020155:3-Hydroxybutyric Acid