Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M, and cloning of the gene.
Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. 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 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.
Nanba
Hirokazu
H
Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan. Hirokazu.Nanba@kaneka.co.jp
Takaoka
Yasuko
Y
Hasegawa
Junzo
J
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019605:Euryarchaeota / Q000201:enzymology* / Q000302:isolation & purification; D005560:Formate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD; D016415:Sequence Alignment
D009243:NAD; D005560:Formate Dehydrogenases