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

从南极耐冷菌冷地黄杆菌KUC-1中分离得到一种依赖NAD~+的乙醇脱氢酶，纯化后总得率约为20%，并对其进行了酶学鉴定。该天然酶表观分子量为160 kDa，由4个相同的40 kDa亚基组成。该酶的等电点为6.7，氧化反应的最适pH为7.0。该酶含有2个锌原子/亚基。该酶只需要NAD^+作为辅酶，对NADH烟酰胺基团的C4位的氢转移具有Pro-R立体专一性。冷地假单胞菌KUC-1乙醇脱氢酶的热稳定性与其耐热性相似，但与其他微生物乙醇脱氢酶的热稳定性不同。该酶在0~85℃的温度范围内活性最强，在70℃时活性最强。计算出60℃时的半衰期为50min，k_&lt；该酶在低温(0-20℃)下(20℃时的kcat/K_m；25,500 mM；-1&gt；min^&lt；-1&gt；)也表现出与嗜冷酶相似的催化效率。乙醇脱氢酶基因全长1,035个核苷酸，编码344个氨基酸残基，相对分子质量为36,823 Da。与Moraxella sp.的氨基酸序列进行了同源性比较。TAE123(67%)、铜绿假单胞菌(65%)和嗜热脂肪杆菌LLD-R(56%)。据我们所知，这是第一个低温活性和耐热酒精脱氢酶的例子。

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.