Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily
Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily
复制标题
DOI：
10.1111/j.1742-4658.2010.07916.x
复制
发表时间：
2010-12
期刊：
影响因子：
--
通讯作者：
K. Yoneda;H. Sakuraba;Ikuo Muraoka;T. Oikawa;T. Ohshima
中科院分区：
文献类型：
--
作者：
K. Yoneda;H. Sakuraba;Ikuo Muraoka;T. Oikawa;T. Ohshima
来自嗜冷细菌冷黄杆菌 KUC-1 的 L-苏氨酸脱氢酶（l-ThrDH；EC 1.1.1.103）的晶体结构与传统的 l-ThrDH 没有序列相似性，在 NAD 和底物类似物甘油存在下测定。不对称单元由通过双重旋转轴相关的两个子单元组成。每个单体由罗斯曼折叠结构域和羧基末端催化结构域组成。 F. frigidimarisl-ThrDH 的整体折叠与 UDP-半乳糖 4-差向异构酶 (GalE) 的整体折叠显着相似；然而，该酶与大肠杆菌和人类 GalE 的结构比较显示，底物和 NAD 结合位点周围的三个环（环 1、环 2 和 NAD 结合环）存在明显的拓扑差异。在 F. frigidimarisl-ThrDH 中，环 1 和 2 插入活性位点空腔，形成阻止 UDP-葡萄糖结合的屏障。或者，环 1 有助于 F. frigidimaris 酶中独特的底物结合袋。 NAD 结合环在大肠杆菌和人类 GalE 中紧密结合 NAD 的腺嘌呤核糖部分，但 F. frigidimarisl-ThrDH 中不存在。因此，与与 GalE 的结合不同，辅因子以可逆的方式与 F. frigidimarisl-ThrDH 结合。底物结合模型表明，反应通过由 Tyr143 驱动并由 Ser118 促进的质子穿梭机制或由 Tyr143 驱动的直接质子转移，通过提取 L-苏氨酸的 β-羟基氢来进行。目前的结构为区分 GalE 样 l-ThrDH 和 GalE 提供了明确的基准。
The crystal structure of a l‐threonine dehydrogenase (l‐ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC‐1, which shows no sequence similarity to conventional l‐ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two‐fold rotation axis. Each monomer consisted of a Rossmann‐fold domain and a carboxyl‐terminal catalytic domain. The overall fold of F. frigidimarisl‐ThrDH showed significant similarity to that of UDP‐galactose 4‐epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD‐binding loop) around the substrate and NAD binding sites. In F. frigidimarisl‐ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP‐glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimarisl‐ThrDH. Consequently, the cofactor binds to F. frigidimarisl‐ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β‐hydroxyl hydrogen of l‐threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE‐like l‐ThrDHs from GalEs.