Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily.
Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily.
复制标题
3-酮基-L-古洛糖酸 6-磷酸脱羧酶（乳清苷 5-单磷酸脱羧酶超家族的成员）催化的反应中存在 1,2-烯二醇中间体的结构证据。
DOI：
10.1021/bi0348819
复制
发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Rayment,Ivan
中科院分区：
文献类型：
--
作者：
Wise,EricL;Yew,WenShan;Gerlt,JohnA;Rayment,Ivan
3-酮基-1-古洛糖酸6-磷酸脱羧酶（KGPDC）和乳清酸核苷5 '-磷酸脱羧酶（OMPDC）是酶超家族（OMPDC超家族）的成员，因为它们是使用不同底物催化机械上不同反应的同源酶。KGPDC催化3-酮基-1-古洛糖酸6-磷酸的Mg 2+离子依赖性脱羧生成1-木酮糖5-磷酸和CO2; OMPDC催化OMP的金属离子非依赖性脱羧生成UMP和CO2。结构研究表明，KGPDC和OMPDC共享几个严格保守的活性位点残基，这些残基被每种酶不同地用于催化其机械上不同的反应。虽然KGPDC催化反应的机理尚未阐明，但认为其通过Mg 2+离子稳定的1，2-烯二醇盐中间体进行。在这里，我们报告的晶体结构的KGPDC复合与l-古洛糖酸6-磷酸，l-苏氨异羟肟酸4-磷酸，和l-木糖醇5-磷酸，类似物的底物，enediolate中间体，和产品，以及与产品，l-木酮糖5-磷酸，在1.2，1.8，1.7，和1.8 μ m分辨率，分别。这些结构支持一种机制，涉及形成的β-1，2-烯二醇中间体。与预期相反，中间体的几何形状不涉及两个烯二醇盐氧原子与Mg 2+离子的双齿配位，而是仅涉及C2上的氧与Mg 2+离子的配位。C1上的氧原子与两个严格保守的活性位点残基Lys 64和Asp 67形成氢键。Lys 64还与C2上的氧相互作用，并可用于稳定1，2-烯二醇盐的顺式构象。这些结构也暗示His 136是使1，2-烯二醇中间体质子化的一般酸。这项研究进一步表明，多个不相关的酶功能可以从一个单一的活性位点架构，而不考虑底物结合亲和力或机制。
3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5‘-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates. KGPDC catalyzes the Mg2+ion-dependent decarboxylation of 3-keto-l-gulonate 6-phosphate to yieldl-xylulose 5-phosphate and CO2; OMPDC catalyzes the metal ion-independent decarboxylation of OMP to UMP and CO2. Structural studies have shown that KGPDC and OMPDC share several strictly conserved active site residues that are used differently by each enzyme to catalyze their mechanistically distinct reactions. Although the mechanism of the KGPDC-catalyzed reaction has yet to be elucidated, it is thought to proceed via a Mg2+ion-stabilized 1,2-enediolate intermediate. Here we report the crystal structures of KGPDC complexed withl-gulonate 6-phosphate,l-threonohydroxamate 4-phosphate, andl-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product,l-xylulose 5-phosphate, at 1.2, 1.8, 1.7, and 1.8 Å resolution, respectively. These structures support a mechanism that involves the formation of acis-1,2-enediolate intermediate. Contrary to expectations, the geometry of the intermediate does not involve bidentate coordination of both enediolate oxygen atoms to the Mg2+ion but rather involves only the coordination of the oxygen on C2 to the Mg2+ion. The oxygen atom on C1 instead forms hydrogen bonds to both Lys64 and Asp67, two strictly conserved active site residues. Lys64 also interacts with the oxygen on C2 and may serve to stabilize a cis conformation of the 1,2-enediolate. These structures also implicate His136 to be the general acid that protonates the 1,2-enediolate intermediate. This study further demonstrates that multiple unrelated enzyme functions can evolve from a single active site architecture without regard for substrate binding affinity or mechanism.