Duewel H S, Radaev S, Wang J, Woodard R W, Gatti D L
The Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2001 Mar 16;276(11):8393-402. doi: 10.1074/jbc.M007884200. Epub 2000 Dec 13.
3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacterium Aquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824-22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd(2+) forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substrate-free enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEP-binding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp(2) to sp(3) hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2(PEP), respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2(PEP), thus triggering the beginning of the catalytic cycle.
嗜热细菌水生栖热菌（Aquifex aeolicus）的3-脱氧-D-甘露糖辛酮酸-8-磷酸合酶（KDO8PS）与其大肠杆菌对应物在活性对二价金属的需求方面存在差异（杜韦尔，H. S.，和伍德尔，R. W.（2000年）《生物化学杂志》275卷，22824 - 22831页）。在此，我们报道了水生栖热菌该酶的晶体结构，其通过以大肠杆菌KDO8PS为模型进行分子置换来确定。在未复合状态以及与磷酸烯醇丙酮酸（PEP）、5-磷酸阿拉伯糖（A5P）和4-磷酸赤藓糖（E4P）的各种组合形成复合物的情况下，测定了该酶无金属和Cd(2+)形式的结构。与大肠杆菌酶一样，水生栖热菌KDO8PS是一种同四聚体，在亚基之间的界面处包含四个不同的活性位点。在无底物的酶中，或者当单独结合A5P或单独结合PEP时，活性位点腔是开放的，而当PEP和A5P（或E4P）一起结合时，它与水相隔离。在有金属存在的情况下，该酶是不对称的，并且似乎在四聚体一侧的活性位点和另一侧的活性位点之间交替催化。在没有金属的情况下，不对称性消失。在这些结构所达到的高分辨率下，可以看到对于催化可能重要的活性位点细节。最值得注意的是，PEP结合口袋的形状迫使PEP在C-2处呈现扭曲的几何形状，这可能预示着在中间体形成过程中从sp(2)到sp(3)杂化的转变，并且可能调节PEP对A5P的反应性。两个水分子分别与C-2(PEP)的si面和re面处于范德华接触。预期蛋白质基团从这两个水分子中的任何一个夺取质子会引发由此产生的氢氧根离子对附近C-2(PEP)的亲核攻击，从而触发催化循环的开始。
