Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis -: Characterization and comparison of the native and the recombinant enzymes

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is;greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C-2 to C-8 hut is also active towards methanol and glycerol and stereospecific for monoterpenes. I hydrothermalis ADH is the first Thermococcale ADN to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.