酵母酒精脱氢酶的低温电子显微镜结构,Biochemistry

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酵母酒精脱氢酶的低温电子显微镜结构
Biochemistry ( IF 2.9 ) Pub Date : 2021-02-23 , DOI: 10.1021/acs.biochem.0c00921
Sai Rohit Guntupalli _{1,

2,

3} , Zhuang Li ₃ , Leifu Chang ₃ , Bryce V Plapp ₄ , Ramaswamy Subramanian ₅

Affiliation

通过X射线晶体学测定的酵母醇脱氢酶的结构表明，亚单位在形成四聚体的两个二聚体的每一个中具有两个不同的构象状态。描述了与催化机理有关的脱辅酶和全酶复合物，但不对称性引发了有关亚基在催化中的协同作用的问题。这项研究使用了冷冻电子显微镜（cryo-EM），为脱辅酶，两种与NADH的二元配合物以及与NAD ⁺和2,2,2-三氟乙醇的三元配合物提供了结构。这些复合物中每一个的所有四个亚基都相同，因为四聚体具有D2对称性，表明不存在预先存在的不对称性，并且亚基可以独立激活。脱辅酶和一种酶-NADH复合物具有“开放”构型，并且催化锌与Cys-43，His-66，Glu-67和Cys-153的配位相反，而另一种酶-NADH复合物和三元复合物具有与锌与Cys-43，His-66，Cys-153以及水或三氟乙醇的氧的经典配位形成闭合构象。构象变化涉及Arg-340与辅酶的焦磷酸基团和Glu-67的相互作用。低温EM和X射线晶体学研究提供了与催化机理相关的结构。

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Cryo-Electron Microscopy Structures of Yeast Alcohol Dehydrogenase

Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers that form the tetramer. Apoenzyme and holoenzyme complexes relevant to the catalytic mechanism were described, but the asymmetry led to questions about the cooperativity of the subunits in catalysis. This study used cryo-electron microscopy (cryo-EM) to provide structures for the apoenzyme, two different binary complexes with NADH, and a ternary complex with NAD⁺ and 2,2,2-trifluoroethanol. All four subunits in each of these complexes are identical, as the tetramers have D2 symmetry, suggesting that there is no preexisting asymmetry and that the subunits can be independently active. The apoenzyme and one enzyme–NADH complex have “open” conformations and the inverted coordination of the catalytic zinc with Cys-43, His-66, Glu-67, and Cys-153, whereas another enzyme–NADH complex and the ternary complex have closed conformations with the classical coordination of the zinc with Cys-43, His-66, Cys-153, and a water or the oxygen of trifluoroethanol. The conformational change involves interactions of Arg-340 with the pyrophosphate group of the coenzyme and Glu-67. The cryo-EM and X-ray crystallography studies provide structures relevant for the catalytic mechanism.

更新日期：2021-03-09

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