Probing the reaction mechanism of IspH protein by x-ray structure analysis
Probing the reaction mechanism of IspH protein by x-ray structure analysis
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DOI：
10.1073/pnas.0913045107
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发表时间：
2010-01-19
影响因子：
11.1
通讯作者：
Groll, Michael
中科院分区：
文献类型：
--
作者：
Graewert, Tobias;Span, Ingrid;Groll, Michael
二磷酸异戊烯基（IPP）和二磷酸二甲基烯基（DMAPP）是类异戊二烯生物合成的两个中心中间体。最近发现的脱氧纤维素5-磷酸途径在其最后一步通过1-羟基-2-甲基-2-丁烯基4-二磷酸还原去羟基化产生IPP和DMAPP的混合物。这种转化是由IspH蛋白催化的，该蛋白在活性位点包含一个中心铁硫簇作为电子转移辅因子。本文报道的IspH与底物、转化底物、产物和PPi复合物的五种晶体结构为该酶的作用机制提供了独特的见解。当IspH蛋白与底物结合在[4Fe-4S]簇上结晶时，IspH与IPP、DMAPP或无机焦磷酸盐配合物的结晶特征为[3Fe-4S]簇。IspH：底物复合物揭示了配体的发夹构象，C(1)羟基与乌头酸酶型[4Fe-4S]簇中的独特位点协调。生成的醇氧化合物配合物与氢键网络耦合，通过Thr167质子继电器充当质子储存库。长时间的x射线照射导致C(1)-O键的断裂（由还原光电子引发）。数据表明，反应机制涉及路易斯酸活化和质子耦合电子转移的结合。所得到的烯丙基自由基中间体可以通过铁硫簇获得第二个电子。反应可以通过一个质子从底物的β -磷酸盐转移到C(1)（产生dapp）或C(3)（产生IPP）而终止。
Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovered deoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH: substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the beta-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).