Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene.
Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.
Nanba
Hirokazu
H
Fine Chemicals Research Laboratories, Kaneka Corporation, Miyamae-machi, Takasago, Hyogo, Japan. Hirokazu.Nanba@kaneka.co.jp
Takaoka
Yasuko
Y
Hasegawa
Junzo
J
D000090:Acetoacetates / Q000494:pharmacology*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004351:Drug Resistance; D005560:Formate Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D021122:Protein Subunits; D013855:Thiobacillus / Q000201:enzymology*
D000090:Acetoacetates; D005561:Formates; D021122:Protein Subunits; D009243:NAD; C030544:formic acid; C111982:ethyl 4-chloro-3-oxobutanoate; D005560:Formate Dehydrogenases