Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: crystallographic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.
Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: crystallographic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.
复制标题
乳清苷5-单磷酸脱羧酶超家族中酶活性的进化：3-酮-L-古洛糖酸6-磷酸脱羧酶活性位点中质子中继系统的晶体学证据。
DOI：
10.1021/bi0497392
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Rayment,Ivan
中科院分区：
文献类型：
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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）是乳清酸核苷单磷酸脱羧酶（OMPDC）超家族的成员，催化3-酮基-1-古洛糖酸6-磷酸甲木酮糖5-磷酸的Mg 2+依赖性脱羧。结构和生物化学证据表明，KGPDC反应通过Mg 2+稳定的1，2-顺式-烯二醇盐中间体进行。烯二醇中间体的质子化以非立体特异性方式发生以形成1-木酮糖5-磷酸。尽管质子传递的确切机制尚不清楚，但Glu 112、His 136和Arg 139已涉及该过程[Yew，W.美国，怀斯，E.，雷蒙岛，和Gerlt，J. A.（2004）Biochemistry 43，6427 - 6437]。令人惊讶的是，这些位置的单个氨基酸取代基本上不降低催化活性，而是改变反应的立体化学过程。在这里，我们报告的X-射线晶体结构的四个突变体，K64 A，H136 A，E112 Q，和E112 Q/H136 A，每一个确定的存在下ofl-苏氨异羟肟酸4-磷酸，一个类似物的enediolate中间体，以1.7，1.9，1.8，和1.9毫微米分辨率，分别。这些结构表明，Lys 64，Glu 112和His 136的取代导致中间体类似物和两个活性位点水分子的位置发生变化，这两个活性位点水分子先前被确定为可能的质子供体。这些变化与所观察到的这些突变体的反应立体化学的改变，从而支持一种反应机制，其中水分子竞争性穿梭质子从侧链的His 136和Arg 139交替面临的thecis-enediolate中间体。这些研究进一步强调了OMPDC超家族中反应机制的广泛变化。
3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC), a member of the orotidine monophosphate decarboxylase (OMPDC) suprafamily, catalyzes the Mg2+-dependent decarboxylation of 3-keto-l-gulonate 6-phosphate tol-xylulose 5-phosphate. Structural and biochemical evidence suggests that the KGPDC reaction proceeds via a Mg2+-stabilized 1,2-cis-enediolate intermediate. Protonation of the enediolate intermediate occurs in a nonstereospecific manner to forml-xylulose 5-phosphate. Although the exact mechanism of proton delivery is not known, Glu112, His136, and Arg139 have been implicated in this process [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004)Biochemistry 43, 6427−6437]. Surprisingly, single amino acid substitutions of these positions do not substantially reduce catalytic activity but rather alter the stereochemical course of the reaction. Here, we report the X-ray crystal structures of four mutants, K64A, H136A, E112Q, and E112Q/H136A, each determined in the presence ofl-threonohydroxamate 4-phosphate, an analogue of the enediolate intermediate, to 1.7, 1.9, 1.8, and 1.9 Å resolution, respectively. These structures reveal that substitutions of Lys64, Glu112, and His136 cause changes in the positions of the intermediate analogue and two active site water molecules that were previously identified as possible proton donors. These changes correlate with the observed alterations in the reaction stereochemistry for these mutants, thereby supporting a reaction mechanism in which water molecules competitively shuttle protons from the side chains of His136 and Arg139 to alternate faces of thecis-enediolate intermediate. These studies further underscore the wide variation in the reaction mechanisms in the OMPDC suprafamily.