NADP-dependent bacterial alcohol dehydrogenases:: Crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii
NADP-dependent bacterial alcohol dehydrogenases:: Crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii
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DOI：
10.1006/jmbi.1998.1750
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发表时间：
1998-05-22
影响因子：
5.6
通讯作者：
Frolow, F
中科院分区：
文献类型：
--
作者：
Korkhin, Y;Kalb, AJ;Frolow, F
我们分别在 2.15 埃和 2.05 埃分辨率下确定了拜氏梭菌 (Clostridium beijerinckii) NADP(H) 依赖性醇脱氢酶 (CBADH) 和空穴酶形式的 X 射线结构，以及热厌氧菌布氏空穴醇脱氢酶 (TBADH) 在 2.5 埃的 X 射线结构。这些是待确定的原核乙醇脱氢酶的第一个结构以及第一个 NADP(H) 依赖性乙醇脱氢酶的结构。 CBADH和TBADH具有75%的序列同一性和非常相似的三维结构。两者都是 222 对称性的四聚体。单体由两个结构域组成：辅因子结合结构域和催化结构域。它们被一个深裂缝分开，在裂缝底部有一个锌原子结合在催化位点上。四聚体由两个二聚体组成，每个二聚体在结构上与脊椎动物的醇脱氢酶的二聚体同源。二聚体通过与域间裂口相对的表面之间的接触形成四聚体，从而使其可从四聚体的表面接近。四聚体封闭了一个具有正表面电位的大内腔。NADP(H) 分子在域间裂缝中与每个单体的辅因子结合域结合。两种细菌醇脱氢酶对 NADP(H) 的特异性由残基 Gly198、Ser199、Arg200 和 Tyr218 决定，后三个与辅因子的 2'-磷酸氧原子形成氢键。 NADP(H) 与 CBADH 结合后，Tyr218 绕 chi(1) 旋转约 120 度，这有利于与腺嘌呤部分的堆积相互作用以及与磷酸氧原子之一的氢键键合，在 apo-CBADH 中，催化锌由残基 Cys37、His59、Asp150 和残基的侧链四配位。 谷氨酸60;在holo-CBADH中，Glu60在四个单体中的三个中从锌上缩回，而在holo-TBADH中，Glu60不参与Zn配位。在两种空穴酶中，但不在猿酶中，残基Ser39和Ser113位于催化锌的第二配位层中。 Asp150的羧基相对于NADP(H)的活性碳取向，从而与pro-S和pro-R氢原子形成氢键。 (C) 1998 学术出版社有限公司。
We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridium beijerinckii (CBADH) in the ape and hole-enzyme forms at 2.15 Angstrom and 2.05 Angstrom resolution, respectively, and of the hole-alcohol dehydrogenase of Thermoanaerobncter brockii (TBADH) at 2.5 Angstrom. These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase. CBADH and TBADH have 75% sequence identity and very similar three-dimensional structures. Both are tetramers of 222 symmetry. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain. These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site. The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential.A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases towards NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor. Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120 degrees about chi(1) which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms, in apo-CBADH the catalytic zinc is tetracoordinated by sidechains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers, whereas in holo-TBADH, Glu60 does not participate in Zn coordination. in both hole-enzymes, but not in the ape-enzyme, residues Ser39 and Ser113 are In the second coordination sphere of the catalytic zinc. The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms. (C) 1998 Academic Press Limited.