Svensson S, Höög J O, Schneider G, Sandalova T
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
J Mol Biol. 2000 Sep 15;302(2):441-53. doi: 10.1006/jmbi.2000.4039.
The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.
小鼠Ⅱ类乙醇脱氢酶（ADH2）与辅酶NADH形成的二元复合物以及与NADH和抑制剂N - 环己基甲酰胺形成的三元复合物的结构已分别确定，分辨率为2.2 Å和2.1 Å。ADH2二聚体在晶体中是不对称的，两个亚基中催化结构域相对于辅酶结合结构域具有不同的取向，导致活性位点裂缝的闭合略有不同。两种构象都大约处于马ADH1的开放脱辅基结构和闭合全酶结构之间的中间位置，因此类似于ADH3的构象。与其他类别的乙醇脱氢酶相比，活性位点裂缝周围的半开放构象和结构差异导致底物结合口袋结构有很大不同，并为醇类和醌类的识别和选择性提供了结构基础。活性位点裂缝比ADH1的更宽大，但不像ADH3的那样开放且呈漏斗状。来自辅酶结合结构域的含有296 - 301位残基的环较短，从而使口袋向辅酶开放。在相对的一侧，含有114 - 121位残基的环伸展跨越结构域间的裂缝。在该环下方形成一个腔，并为底物结合口袋增加了一个附属部分。Asp301位于口袋入口处，可能控制ω - 羟基脂肪酸的结合，ω - 羟基脂肪酸作为抑制剂而非底物。小鼠ADH2是一种低效的ADH，其氢转移步骤较慢。通过将Pro47替换为His，乙醇脱氢酶活性得以恢复。在此，该P47H突变体与NADH复合物的结构已确定，分辨率为2.5 Å。His47的位置合适，可作为底物去质子化的催化碱。此外，在更闭合的亚基中，辅酶被允许处于更靠近催化锌的位置。这与来自醇盐中间体的氢转移一致，其中Pro/His替换聚焦于酶的功能。
