Mechanistic studies of IspH in the deoxyxylulose phosphate pathway:: Heterolytic C-O bond cleavage at C4 position
Mechanistic studies of IspH in the deoxyxylulose phosphate pathway:: Heterolytic C-O bond cleavage at C4 position
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DOI：
10.1021/ja710245d
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发表时间：
2008-02-20
影响因子：
15
通讯作者：
Liu, Pinghua
中科院分区：
文献类型：
--
作者：
Xiao, Youli;Zhao, Zongbao K.;Liu, Pinghua
类异戊二烯是自然界中最大和结构最多样化的代谢物组之一。它们的生物合成需要两个前体，异戊烯基二磷酸（IPP）和它的异构体，二甲基烯丙基二磷酸（DMAPP）。IPP和DMAPP的合成有两种不同的途径：磷酸脱氧木酮糖（DXP）途径和甲羟戊酸（MVA）途径。更重要的是，这两条途径在不同的王国中有明确的分布。大多数病原菌和原生动物寄生虫利用DXP途径，而动物通过MVA途径从乙酰辅酶A合成其类异戊二烯前体。植物具有DXP和MVA途径。因此，对DXP途径酶的机制研究可能会导致开发基于机制的抑制剂，如除草剂，广谱抗生素和抗疟疾药物。DXP途径中的IspH催化（E）-4-羟基-3-甲氧基-2-丁烯基二磷酸（HMBPP）的还原脱水以形成IPP和DMAPP，这是DXP途径中的最后一步。最近文献报道的EPR研究表明，IspH是一种独特的含铁位点的[4Fe-4S]蛋白。在这项研究中，我们使用两种底物类似物研究了IspH催化的还原脱水机制。本文报道的数据提供了证据，不仅支持反应期间C1位C-O键的完整性，它们还表明在IspH催化的还原脱水反应中，C4位的异裂解C-O键断裂。我们的动力学研究还表明，C4羟基参与底物结合。由于IspH催化的还原脱水反应不属于两类已知的独特的含铁位点的[4Fe-4S]蛋白，乌头酸酶型和自由基SAM型酶，IspH可能代表一类新的含铁硫蛋白。
Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. Their biosyntheses require two precursors, isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). There are two different pathways for the synthesis of IPP and DMAPP: the deoxyxylulose phosphate (DXP) pathway and the mevalonic acid (MVA) pathway. More importantly, these two pathways have a well-defined distribution among different kingdoms. Most pathogenic bacteria and protozoan parasites utilize the DXP pathway, while animals synthesize their isoprenoid precursors from acety-CoA via the MVA pathway. Plants have both DXP and MVA pathways. Thus, mechanistic studies on the DXP pathway enzymes may lead to the development of mechanism-based inhibitors as herbicides, broad-spectrum antibiotics, and antimalaria drugs. IspH in the DXP pathway catalyzes the reductive dehydration of (E)-4-hydroxy-3-metho-2-butenyl diphosphate (HMBPP) to form IPP and DMAPP, the last step in the DXP pathway. Recent EPR studies reported in literature suggest that IspH is a unique iron-site-containing [4Fe-4S] protein. In this study, we studied the IspH-catalyzed reductive dehydration mechanism using two substrate analogues. Our data reported herein provide evidence to not only support the integrity of the C1 position C-O bond during reaction, they also suggest a heterolytic C-O bond cleavage at the C4 position for IspH-catatyzed reductive dehydration reaction. Our kinetic studies also suggest that the C4 hydroxyl group is involved in substrate binding. Because the IspH-catalyzed reductive dehydration reaction does not fall into the two known classes of unique iron-site-containing [4Fe-4S] proteins, aconitase-type and radical SAM-type enzymes, IspH may represent a new class of iron-sulfur-containing proteins.