The Stereochemical Course and Mechanism of the IspH Reaction
The Stereochemical Course and Mechanism of the IspH Reaction
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DOI：
10.1002/anie.201201110
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Dickschat, Jeroen S.
中科院分区：
文献类型：
--
作者：
Citron, Christian A.;Brock, Nelson L.;Dickschat, Jeroen S.
萜烯单体二甲基烯丙基二磷酸（DMAPP）和异戊烯基二磷酸（IPP）的生物合成存在两种途径：甲羟戊酸途径[1]和脱氧木酮糖磷酸（DOX）途径。[2，3]许多病原菌和疟疾寄生虫恶性疟原虫专门使用DOX途径，这在这些生物体中至关重要。[4，5]由于这种途径在人类中不存在，因此其酶代表了新抗菌药物的有吸引力的靶标。[6]因此，对酶机制的详细了解具有很高的兴趣。DOX途径的中间体和酶都已被鉴定（示意图1）。[7]特别地，IspH催化1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸酯（HMBPP，7）转化为DMAPP（8）和IPP（9）的约1：5混合物的独特机理已经引起了相当大的兴趣，并且仍在讨论中。IspH的晶体结构显示中心Fe 3S 4簇，[8]而其他数据则支持Fe 4S 4簇。[9]最近，IspH在其衬底7存在下的晶体结构被细化为具有Fe 4S 4簇的结构。[10]第四个不稳定的铁中心结合底物7，[9d，10]以及有效的抑制剂，如吡啶二磷酸盐和炔。[11]无底物酶的穆斯堡尔参数表明，该Fe中心与三个S和两个或三个其他配体（O或N）配位，这些配体在与底物结合时解离。[9b已经提出了IspH反应的三种机制（方案2）。机制A，由Rohdich等人提出。（方案2A）类似于配合物10的Birch还原，通过单电子转移、质子化和消除水至烯丙基自由基11，以及第二电子转移以得到烯丙基阴离子12。其在C-2或C-4的质子化分别导致IPP和DMAPP。[12]这一机制得到了底物类似物同位素标记实验的支持。[13]机制B，由Wang等人提出。（方案2B）基于捕获在非反应性E126 A突变体中的顺磁性中间体的ENDOR光谱检测，其被解释为金属环丙烷物质。[14]或者，该中间体也可以描述为η2-烯基π络合物。所描述的机理从10开始，通过单电子还原形成η2-烯基/金属配合物13，然后质子化/脱水得到η1-烯丙基配合物14。第二次电子转移产生12作为IPP和DMAPP的前体。机理C由Altincicek等人提出，并且包括由金属中心的刘易斯酸性辅助的10的初始质子化/脱水以产生烯丙基阳离子15，其经历经由11至12的两个还原步骤和随后的质子化以提供IPP和DMAPP。[15]这一机制与底物类似物中吸电子取代基的次要作用相矛盾。[16]所有三种机制的共同中间体是初始络合物10和烯丙基阴离子12。IPP和DMAPP的产物比被认为是由HMBPP的末端磷酸基团的质子转移控制的，其中两个氧原子与C-1和C-3的距离为3.4-3.5（方案2A）。[10该假设与观察到的HMBPP从Si面的立体特异性C-3质子化完全一致。[17]DOX途径的几乎所有步骤的立体化学过程已经被研究。[18]沿着这一途径，1的C-3氢原子（HA，方案1）变成2中的醛氢原子，并最终成为3中C-1处的pro-S氢原子，而pro-R氢原子（HC）是.
Two pathways exist for the biosynthesis of the terpene monomers dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP): the mevalonate pathway [1] and the deoxyxylulose phosphate (DOX) pathway.[2, 3] Many pathogenic bacteria and the malaria parasite Plasmodium falciparum exclusively use the DOX pathway, which is essential in these organisms.[4, 5] Since this pathway is not present in humans, its enzymes represent attractive targets for new antimicrobial drugs.[6] Therefore, a detailed understanding of the enzyme mechanisms is of high interest. The intermediates and enzymes of the DOX pathway have all been identified (Scheme1).[7] In particular, the unique mechanism of IspH that catalyzes the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP, 7) into an approximate 1: 5 mixture of DMAPP (8) and IPP (9) has attracted considerable interest and is still under discussion. The crystal structure of IspH showed a central Fe3S4 cluster,[8] while other data were in favor of an Fe4S4 cluster.[9] Recently, the crystal structure of IspH in the presence of its substrate 7 was refined to a structure with an Fe4S4 cluster.[10] The fourth labile iron center binds the substrate 7,[9d, 10] as well as potent inhibitors such as pyridine diphosphates and alkynes.[11] Mçssbauer parameters of the substrate-free enzyme suggested that this Fe center is coordinated by three S and two or three other ligands (O or N) that dissociate upon substrate binding.[9b, d]Three mechanisms have been proposed for the IspH reaction (Scheme 2). Mechanism A, suggested by Rohdich et al.(Scheme 2A), resembles a Birch reduction of complex 10 by a one-electron transfer, protonation, and elimination of water to the allyl radical 11, and a second electron transfer to give the allyl anion 12. Its protonation at C-2 or C-4 results in IPP and DMAPP, respectively.[12] This mechanism is supported by isotopic labeling experiments with substrate analogues.[13] Mechanism B, proposed by Wang et al.(Scheme 2B), is based on the ENDOR spectroscopic detection of a paramagnetic intermediate trapped in the unreactive E126A mutant that was interpreted as a metallacyclopropane species.[14] Alternatively, this intermediate may also be described as an η2-alkenyl π complex. The delineated mechanism proceeds from 10 by one-electron reduction to form the η2-alkenyl/metallacycle 13 followed by a protonation/dehydration to give the η1-allyl complex 14. A second electron transfer yields 12 as the precursor for IPP and DMAPP. Mechanism C was proposed by Altincicek et al. and includes the initial protonation/dehydration of 10 assisted by the Lewis acidity of the metal center to generate the allyl cation 15, which undergoes two reduction steps via 11 to 12 and a subsequent protonation to afford IPP and DMAPP.[15] This mechanism is contradicted by the minor effect of electron-withdrawing substituents in substrate analogues.[16] Common intermediates of all three mechanisms are the initial complex 10 and the allyl anion 12. The product ratio of IPP and DMAPP was suggested to be controlled by the proton transfer from the terminal phosphate group of HMBPP, where the two oxygen atoms are at a distance of 3.4–3.5 to C-1 and C-3 (Scheme 2A).[10, 12b] This hypothesis is in full agreement with the observed stereospecific C-3 protonation of HMBPP from the Si face.[17] The stereochemical course of almost all the steps of the DOX pathway have been studied.[18] Along this pathway, the C-3 hydrogen atom of 1 (HA, Scheme 1) becomes the aldehyde hydrogen atom in 2 and ends up as the pro-S hydrogen atom at C-1 in 3, whereas the pro-R hydrogen atom (HC) is …