Bioscience, Biotechnology, and Biochemistry 2003-10-01

Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene.

Hirokazu Nanba, Yasuko Takaoka, Junzo Hasegawa

Index: Biosci. Biotechnol. Biochem. 67(10) , 2145-53, (2003)

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Abstract

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.