Catalytic mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase in complex with sulfate, magnesium, and triton
Catalytic mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase in complex with sulfate, magnesium, and triton
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DOI：
10.1074/jbc.m302687200
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发表时间：
2003-08-01
影响因子：
4.8
通讯作者：
Wang, AHJ
中科院分区：
文献类型：
--
作者：
Chang, SY;Ko, TP;Wang, AHJ
十一异戊烯焦磷酸合酶（UPPs）催化焦磷酸法呢酯（FPP）通过与八种异戊烯焦磷酸（IPP）缩合而链延长成十一异戊烯焦磷酸（UPP）。大肠杆菌的UPPs是一个二聚体，每个亚基由253个氨基酸残基组成。产物的链长由疏水活性位点隧道调节。本文对E. coliUPPs被精细化到1.73埃分辨率，其显示结合的硫酸根和镁离子以及Triton X-100分子。氨基酸残基72 - 82，其包含在先前的脱辅基酶结构中未见的必需催化环（Ko，T. P.，Chen，Y. K.，罗宾逊，H.，蔡培中，高氏G.，Chen，中国山核桃A. P. - C.的方法，Wang，中国山核桃A. H.- J.，Liang，P.- H.（2001）J.Biol.Chem.276，47474 - 47482），在一个亚基中也变得可见。硫酸根离子表明FPP和IPP的焦磷酸基团在活性位点中的位置。Mg 2+被来自不同亚基的His- 199和Glu-213螯合，并且可能起结构作用而不是催化作用。而金属离子位于IPP结合位点附近，His- 199和Glu-213双突变为丙氨酸后，IPP的Km值显著增加。在隧道内，一个海卫一围绕着隧道的顶部，另一个占据着底部。这两个Triton分子可以模拟活性位点中UPP产物的烃部分。动力学分析表明，高浓度（> 1%）的Triton抑制酶活性。
Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPPs from Escherichia coli is a dimer, and each subunit consists of 253 amino acid residues. The chain length of the product is modulated by a hydrophobic active site tunnel. In this paper, the crystal structure of E. coli UPPs was refined to 1.73 Angstrom resolution, which showed bound sulfate and magnesium ions as well as Triton X-100 molecules. The amino acid residues 72 - 82, which encompass an essential catalytic loop not seen in the previous apoenzyme structure ( Ko, T.- P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y.- G., Chen, A. P.- C., Wang, A. H.- J., and Liang, P.- H. ( 2001) J. Biol. Chem. 276, 47474 - 47482), also became visible in one subunit. The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site. The Mg2+ is chelated by His- 199 and Glu-213 from different subunits and possibly plays a structural rather than catalytic role. However, the metal ion is near the IPP-binding site, and double mutation of His- 199 and Glu-213 to alanines showed a remarkable increase of K-m value for IPP. Inside the tunnel, one Triton surrounds the top portion of the tunnel, and the other occupies the bottom part. These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site. Kinetic analysis indicated that a high concentration (> 1%) of Triton inhibits the enzyme activity.