Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate - Roles of the metal ion and conserved residues in catalysis
Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate - Roles of the metal ion and conserved residues in catalysis
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DOI：
10.1074/jbc.m502121200
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发表时间：
2005-05-27
影响因子：
4.8
通讯作者：
Liang, PH
中科院分区：
文献类型：
--
作者：
Guo, RT;Ko, TP;Liang, PH
十一异戊二烯焦磷酸合酶（UPPs）催化法尼基焦磷酸（FPP）与八个异戊烯基焦磷酸（IPP）连续缩合反应，形成新的顺式双键，生成十一异戊二烯焦磷酸，作为细菌细胞壁肽聚糖合成的脂质载体。先前确定了大肠杆菌 UPP 的结构，其为作为脱辅基酶的斜方晶体形式，与 Mg2+/硫酸盐/Triton 复合，并与结合的 FPP 复合。在进一步研究其催化机制时，野生型 UPP 和 D26A 突变体在新的三角晶胞中结晶，其中 Mg2+/IPP/法尼基硫代焦磷酸盐（一种 FPP 类似物）与活性位点结合。在野生型酶中，Mg2+ 由法呢基硫代焦磷酸盐的焦磷酸盐、Asp26 的羧酸盐和三个水分子配位。在突变酶中，它与 IPP 的焦磷酸结合。 UPP 催化速率的 [Mg2+] 依赖性表明，当 [Mg2+] = 1 mM 时活性最大，但当 Mg2+ 离子过量 (50 mm) 时活性显着下降。如果没有 Mg2+，IPP 仅在高浓度时才能与 UPP 结合。 Asp26 突变为其他带电氨基酸会导致 UPP 活性显着降低。 Asp26 的作用可能是协助 Mg2+ 从 IPP 迁移到 FPP，从而通过 FPP 的焦磷酸基团电离来引发缩合反应。其他保守残基，包括 His(43)、Ser(71)、Asn(74) 和 Arg(77)，可用作通用酸/碱和焦磷酸盐载体。我们的结果显着提高了对 UPP 酶反应的理解。
Undecaprenyl pyrophosphate synthase ( UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg2+/sulfate/ Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg2+/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg2+ is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp26, and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg2+] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg2+] = 1 mM but drops significantly when Mg2+ ions are in excess (50 mm). Without Mg2+, IPP binds to UPPs only at high concentration. Mutation of Asp26 to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp26 is probably to assist the migration of Mg2+ from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.