Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp KUC-1 -: Purification, molecular characterization and expression
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp KUC-1 -: Purification, molecular characterization and expression
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DOI：
10.1046/j.1432-1327.2001.02353.x
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发表时间：
2001-08-01
期刊：
影响因子：
--
通讯作者：
Soda, K
中科院分区：
文献类型：
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作者：
Oikawa, T;Yamanaka, K;Soda, K
我们在嗜冷菌Cytophaga sp.的细胞提取液中发现了Valine脱氢酶。Kuc-1，从南极海水中分离并纯化了该酶。凝胶过滤测得该酶的相对分子质量约为154 kDa，SDS/PAGE测得其亚基的相对分子质量为43 kDa，表明该酶为同源四聚体。该酶需要NAD(+)作为辅酶，催化L-缬氨酸、L-异亮氨酸、L-亮氨酸的氧化脱氨反应和α-酮基异戊酸酯、α-酮基戊酸酯、α-酮基异戊酸酯和α-酮基己酸酯的还原胺化反应。该反应通过等序的bi-bi机理进行。该酶对热处理高度敏感，在45℃时的半衰期为2.4min。L-缬氨酸和NAD(+)在20℃时的k(CAT)/K-m(穆穆(-1)·s(-1))值分别为27.48和421.6。该酶对辅酶烟酰胺基团的C4位上的氢转移具有前S立体专一性。将Valine脱氢酶基因克隆到大肠杆菌Novablue中，根据编码该酶基因的核苷酸序列推测了该酶的一级结构。该酶由370个氨基酸残基组成，与天蓝色链球菌和德国链球菌的同源性分别为46.7%和43.1%。Cytophaga sp.与其他ValDHs相比，Kuc-1ValDHs含有更少的脯氨酸和精氨酸残基。这些变化可能导致了细胞吞噬酶分子构象灵活性的增加，从而增强了低温下的催化活性。
We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be approximate to 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD(+) as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha -ketoisovalerate, alpha -ketovalerate, alpha -ketoisocaproate, and alpha -ketocaproate. The reaction proceeds through an iso-ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 degreesC was estimated to be 2.4 min. The k(cat)/K-m (mu mu (-1).s(-1)) values for L-valine and NAD(+) at 20 degreesC were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.