CLONING OF FORMATE DEHYDROGENASE GENE FROM A METHANOL-UTILIZING BACTERIUM MYCOBACTERIUM-VACCAE N-10

The gene of NAD(+)-dependent formate dehydrogenase (FDH) from Mycobact erium vaccae N10 was cloned into Escherichia coli by hybridization wit h digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain rea ction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different o rganisms. An open-reading frame of 1200 bp exhibited extremely high se quence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was i dentical to that of Pseudomonas sp. 101 (PsFDH) except for two amino a cid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lys ine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate- 61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by s ite-directed mutagenesis. All the mutant enzymes showed higher thermos tability than the wildtype McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.