Strategy for cold adaptation of enzyme : studies on structure and molecular evolution of cold active alcohol dehydrogenase

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An NAD^+-dependent alcohol dehydrogenase from the Antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1 was purified to be homogeneity with an overall yield of about 20% and characterized enzymologically. The native enzyme had an apparent molecular mass of 160 kDa and consisted of four identical 40 kDa subunits. The pI of the enzyme was determined to be 6.7 and its optimum pH for oxidation reaction was 7.0. The enzyme contained 2 zinc atoms/subunit. The enzyme exclusively required NAD^+ as a coenzyme and showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. F.frigidimaris KUC-1 alcohol dehydrogenase showed a remarkable thermal stability similar to its thermophilic counterparts and in contrast to other microbial alcohol dehydrogenases. The enzyme was active in the temperature range of 0 to over 85℃ and the most active at 70℃. The half-life time and k_<cat> at 60℃ were calculated to be 50 min and 27,370 (min^<-1>), respectively. The enzyme also showed high catalytic efficiency at low temperatures (0-20℃) (kcat/K_m at 20℃ ; 25,500 mM^<-1> min^<-1>) similar to its psychrophilic counterpart. The alcohol dehydrogenase gene was composed of 1,035 bp and coded 344 amino acid residues with an estimated molecular mass of 36,823 Da. The sequence identities were found with the amino acid sequences of Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%), and Geobacillus stearothermophilus LLD-R (56%) alcohol dehydrogenases. To our knowledge, this is the first example of a cold-active and thermostable alcohol dehydrogenase.