Auerbach G, Ostendorp R, Prade L, Korndörfer I, Dams T, Huber R, Jaenicke R
Max-Planck-Institut für Biochemie Abt. Strukturforschung, 82152, Martinsried, Germany.
Structure. 1998 Jun 15;6(6):769-81. doi: 10.1016/s0969-2126(98)00078-1.
L(+)-Lactate dehydrogenase (LDH) catalyzes the last step in anaerobic glycolysis, the conversion of pyruvate to lactate, with the concomitant oxidation of NADH. Extensive physicochemical and structural investigations of LDHs from both mesophilic and thermophilic organisms have been undertaken in order to study the temperature adaptation of proteins. In this study we aimed to determine the high-resolution structure of LDH from the hyperthermophilic bacterium Thermotoga maritima (TmLDH), the most thermostable LDH to be isolated so far. It was hoped that the structure of TmLDH would serve as a model system to reveal strategies of protein stabilization at temperatures near the boiling point of water.
The crystal structure of the extremely thermostable TmLDH has been determined at 2.1 A resolution as a quaternary complex with the cofactor NADH, the allosteric activator fructose-1,6-bisphosphate, and the substrate analog oxamate. The structure of TmLDH was solved by Patterson search methods using a homology-based model as a search probe. The native tetramer shows perfect 222 symmetry. Structural comparisons with five LDHs from mesophilic and moderately thermophilic organisms and with other ultrastable enzymes from T. maritima reveal possible strategies of protein thermostabilization.
Structural analysis of TmLDH and comparison of the enzyme to moderately thermophilic and mesophilic homologs reveals a strong conservation of both the three-dimensional fold and the catalytic mechanism. Going from lower to higher physiological temperatures a variety of structural differences can be observed: an increased number of intrasubunit ion pairs; a decrease of the ratio of hydrophobic to charged surface area, mainly caused by an increased number of arginine and glutamate sidechains on the protein surface; an increased secondary structure content including an additional unique 'thermohelix' (alphaT) in TmLDH; more tightly bound intersubunit contacts mainly based on hydrophobic interactions; and a decrease in both the number and the total volume of internal cavities. Similar strategies for thermal adaptation can be observed in other enzymes from T. maritima.
L(+)-乳酸脱氢酶（LDH）催化无氧糖酵解的最后一步，即丙酮酸转化为乳酸，并伴随NADH的氧化。为了研究蛋白质的温度适应性，已经对嗜温生物和嗜热生物的LDH进行了广泛的物理化学和结构研究。在本研究中，我们旨在确定来自嗜热细菌海栖热袍菌（TmLDH）的LDH的高分辨率结构，TmLDH是迄今为止分离出的最耐热的LDH。人们希望TmLDH的结构能够作为一个模型系统，揭示在接近水沸点的温度下蛋白质稳定化的策略。
已确定极端耐热的TmLDH的晶体结构，分辨率为2.1埃，其为与辅因子NADH、变构激活剂果糖-1,6-二磷酸和底物类似物草氨酸盐形成的四级复合物。使用基于同源性的模型作为搜索探针，通过帕特森搜索方法解析了TmLDH的结构。天然四聚体呈现完美的222对称性。与来自嗜温生物和中度嗜热生物的五种LDH以及来自海栖热袍菌的其他超稳定酶的结构比较揭示了蛋白质热稳定化的可能策略。
TmLDH的结构分析以及该酶与中度嗜热和嗜温同源物的比较揭示了三维折叠和催化机制的高度保守性。从较低生理温度到较高生理温度，可以观察到多种结构差异：亚基内离子对数量增加；疏水与带电表面积之比降低，主要是由于蛋白质表面精氨酸和谷氨酸侧链数量增加；二级结构含量增加，包括TmLDH中额外独特的“热螺旋”（αT）；亚基间接触更紧密，主要基于疏水相互作用；内部腔的数量和总体积减少。在海栖热袍菌的其他酶中也可以观察到类似的热适应策略。
