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.
Fine Chemicals Research Laboratories, Kaneka Corporation, Miyamae-machi, Takasago, Hyogo, Japan. Hirokazu.Nanba@kaneka.co.jp
Hirokazu
H
Nanba
Yasuko
Y
Takaoka
Junzo
J
Hasegawa
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