Ishiyama Noboru, Creuzenet Carole, Miller Wayne L, Demendi Melinda, Anderson Erin M, Harauz George, Lam Joseph S, Berghuis Albert M
Department of Biochemistry and Department of Microbiology and Immunology, McGill University, Montreal, Quebec.
J Biol Chem. 2006 Aug 25;281(34):24489-95. doi: 10.1074/jbc.M602393200. Epub 2006 May 1.
FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 A. These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of "real time" enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.
来自人类病原体幽门螺杆菌的FlaA1是一种参与糖类生物合成的酶，已被证明对致病性至关重要。在此，我们展示了FlaA1在存在底物、抑制剂和结合辅因子的情况下的五个晶体结构，分辨率范围为2.8至1.9埃。这些结构表明该酶是短链脱氢酶/还原酶超家族的一个新成员。额外的电子显微镜研究表明该酶具有六聚体甜甜圈形状的四级结构。对“实时”酶 - 底物反应的核磁共振分析表明FlaA1是一种UDP - GlcNAc反转4,6 - 脱水酶，这表明该酶催化假氨基糖酸衍生物生物合成途径的第一步，假氨基糖酸衍生物与蛋白质糖基化有关。在定点诱变和计算模拟证据的指导下，提出了一种三步反应机制，其中Lys - 133同时作为催化酸和碱发挥作用。
