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

酶的冷适应策略：冷活性乙醇脱氢酶的结构和分子进化研究

基本信息

批准号：
16550150
负责人：
OIKAWA Tadao
金额：
$ 1.73万
依托单位：
Kansai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

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.

从南极耐冷菌Flavobacterium frigidimaris KUC-1中分离纯化了一种NAD^+依赖型乙醇脱氢酶，总收率约20%，并对其进行了酶学性质的研究。天然酶的表观分子量为160 kDa，由4个相同的40 kDa亚基组成。该酶的pI为6.7，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，并在NADH烟酰胺部分的C4位置上表现出pro-R立体专一性的氢转移。F.frigidimaris KUC-1乙醇脱氢酶显示出与其嗜热对应物相似的显著的热稳定性，并且与其他微生物乙醇脱氢酶形成对比。该酶在0 ~ 85℃以上的温度范围内均有活性，70℃时活性最强。60℃下的半衰期和k_<cat>分别计算为50 min和27，370（min^<-1>）。该酶在低温（0-20℃）下也显示出与其嗜冷对应物相似的高催化效率（kcat/K_m在20℃ ; 25，500 mM·<-1>min·<-1>）。该基因全长1,035 bp，编码344个氨基酸，分子量为36,823 Da，与莫拉氏菌TAE 123（67%）、铜绿假单胞菌（65%）和嗜热脂肪土芽孢杆菌LLD-R（56%）的乙醇脱氢酶氨基酸序列一致。据我们所知，这是第一个冷活性和热稳定的醇脱氢酶的例子。