Kazuoka Takayuki, Takigawa Shouhei, Arakawa Noriaki, Hizukuri Yoshiyuki, Muraoka Ikuo, Oikawa Tadao, Soda Kenji
Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka-fu 564-8680, Japan.
J Bacteriol. 2003 Aug;185(15):4483-9. doi: 10.1128/JB.185.15.4483-4489.2003.
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 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. 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 bp 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.
从南极海水中分离出了一种嗜冷细菌——噬纤维菌属菌株KUC-1，该菌株能大量产生一种依赖NAD⁺的L-苏氨酸脱氢酶，并对该酶进行了纯化。该酶的分子量估计为139,000，亚基分子量确定为35,000。该酶是同四聚体。原子吸收分析表明该酶不含金属。在这些方面，噬纤维菌的这种酶与迄今研究过的其他L-苏氨酸脱氢酶不同。L-苏氨酸和DL-苏式-3-羟基正缬氨酸是底物，NAD⁺及其一些类似物作为辅酶。该酶在pH 9.5和45℃时表现出最大活性。酶的动力学参数受温度影响很大。L-苏氨酸的Kₘ在20℃时最低。用丙酮酸和腺苷-5'-二磷酸核糖进行的终产物抑制研究表明，该酶反应通过有序的双底物双产物机制进行，其中NAD⁺在L-苏氨酸之前与酶结合，2-氨基-3-氧代丁酸在NADH之前从酶中释放。该酶基因被克隆到大肠杆菌中，并对其核苷酸进行了测序。该酶基因包含一个939 bp的开放阅读框，编码一个由312个氨基酸残基组成的蛋白质。该酶的氨基酸序列与金黄色葡萄球菌的UDP-葡萄糖4-表异构酶的氨基酸序列有显著相似性，属于短链脱氢酶-还原酶超家族。相比之下，大肠杆菌的L-苏氨酸脱氢酶属于中链醇脱氢酶家族，其氨基酸序列与噬纤维菌的酶完全不相似。L-苏氨酸脱氢酶与一种表异构酶有显著相似性，这是首次被证明。在大肠杆菌和人UDP-葡萄糖4-表异构酶催化中起重要作用的氨基酸残基在噬纤维菌的酶中高度保守，除了参与底物结合的残基。
