Purification and sequence analysis of a novel NADP(H)-dependent type III alcohol dehydrogenase from Thermococcus strain AN1.

Abstract

An NADP(H)-dependent alcohol dehydrogenase was isolated from the hyperthermophilic archaeon Thermococcus strain AN1. This enzyme is a homotetramer with a subunit molecular weight of 46,700. The enzyme oxidizes a series of primary linear alcohols but not methanol. The pH and temperature optima with ethanol as the substrate are 6.8 to 7.0 and 85 degrees C, respectively. The enzyme readily reduced acetaldehyde with NADPH as the cofactor. The gene encoding this enzyme has been cloned and sequenced. An open reading frame of 1,218 bp, starting with ATG and ending with TGA, was identified and corresponded to 406 amino acids. Sequence comparisons show that this Thermococcus strain AN1 enzyme has significant homologies with enzymes from the newly defined type III alcohol dehydrogenase family. Thermococcus strain AN1 alcohol dehydrogenase is the first archaeal enzyme belonging to this family.