Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp strain KUC-1
Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp strain KUC-1
复制标题
DOI：
10.1128/jb.185.15.4483-4489.2003
复制
发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Soda, K
中科院分区：
文献类型：
--
作者：
Kazuoka, T;Takigawa, S;Soda, K
嗜冷菌Cytophaga sp.从南极海水中分离到一株能大量产生依赖NAD+的L苏氨酸脱氢酶的菌株KUC-1，并对该酶进行了纯化。该酶的相对分子质量为139,000，亚基的相对分子质量为35,000。这种酶是一种同源四聚体。原子吸收分析表明，该酶不含金属。在这些方面，细胞吞噬酶不同于迄今为止研究过的其他L-苏氨酸脱氢酶。以L-苏氨酸和DL-苏氨酸-3-羟基去甲丙氨酸为底物，NAD(+)及其类似物为辅酶。该酶最适pH为9.5，最适温度为45℃。酶的动力学参数受温度的影响较大。L-苏氨酸的K-m在20℃时最低。丙酮酸和腺苷-5‘-二磷酸核糖的末端抑制研究表明，酶反应遵循有序的BiBi键，NAD(+)在NADH之前与酶结合，2-氨基-3-氧丁酸在NADH之前从酶中释放。将该酶基因克隆到大肠杆菌中，并进行核苷酸序列测定。该酶基因编码312个氨基酸残基，开放阅读框为939。该酶的氨基酸序列与金黄色葡萄球菌的UDP-葡萄糖4-差向异构酶有很大的相似性，属于短链脱氢酶-还原酶超家族。相比之下，来自大肠杆菌的L-苏氨酸脱氢酶属于中链酒精脱氢酶家族，其氨基酸序列与细胞吞噬酶完全不相似。L-苏氨酸脱氢酶与同分异构体有显著的相似性，这是首次发现的。除了参与底物结合的氨基酸残基外，在大肠杆菌和人UDP-葡萄糖4-差向异构酶的催化作用中起重要作用的氨基酸残基在细胞吞噬酶中高度保守。
A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degreesC. The kinetic parameters of the enzyme are highly influenced by temperatures. The K-m for L-threonine was lowest at 20 degreesC. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 by encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.