Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
J Am Chem Soc. 2010 Oct 20;132(41):14509-20. doi: 10.1021/ja101764w.
(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE/IspG) converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the methyl-erythritol phosphate (MEP) pathway for isoprene biosynthesis. MEcPP is a cyclic compound and the reaction involves the opening of the ring and removal of the C3 hydroxyl group consuming a total of two electrons. The enzyme contains a single [4Fe-4S] cluster in its active site. Several paramagnetic species are observed in steady-state and pre-steady-state kinetic studies. The first signal detected is from a transient species that displays a rhombic electron paramagnetic resonance (EPR) signal with g(xyz) = 2.000, 2.019, and 2.087 (FeS(A)). A second set of EPR signals (FeS(B)) accumulated during the reaction. Labeling studies with (57)Fe showed that all species observed are iron-sulfur-based. (31)P-ENDOR measurements on the FeS(A) species showed a weak (31)P coupling which is in line with binding of the substrate to the enzyme in close proximity of the active-site cluster. On the basis of the EPR/ENDOR measurements, we propose a direct binding of the substrate to the [4Fe-4S] cluster during the reaction, and therefore that the iron-sulfur cluster is directly involved in a reductive elimination of a hydroxyl group. The FeS(B) signal also showed (31)P coupling; in this case, however, it could be shown that the signal is due to the binding of the reaction product HMBPP to the active site cluster.
(E)-4-羟基-3-甲基-2-丁烯基二磷酸合酶（GcpE/IspG）在异戊二烯生物合成的甲基赤藓醇磷酸（MEP）途径的倒数第二步中将 2-C-甲基-D-赤藓醇-2,4-环二磷酸（MEcPP）转化为（E）-4-羟基-3-甲基-2-丁烯基二磷酸（HMBPP）。MEcPP 是一种环状化合物，该反应涉及开环和去除 C3 羟基，总共消耗两个电子。该酶在其活性位点含有一个单一的[4Fe-4S]簇。在稳态和预稳态动力学研究中观察到几种顺磁物质。检测到的第一个信号来自于一种瞬态物质，该物质显示出具有 rhombic 电子顺磁共振（EPR）信号，g(xyz) = 2.000、2.019 和 2.087（FeS(A)）。在反应过程中积累了第二组 EPR 信号（FeS(B)）。用（57）Fe 进行的标记研究表明，所有观察到的物质都是铁硫基。对 FeS(A)物种的（31）P-ENDOR 测量显示出较弱的（31）P 偶合，这与底物在靠近活性位点簇的位置与酶的紧密结合一致。基于 EPR/ENDOR 测量结果，我们提出了在反应过程中底物直接与[4Fe-4S]簇结合，因此铁硫簇直接参与羟基的还原消除。FeS(B)信号也显示出（31）P 偶合；然而，在这种情况下，可以证明该信号是由于反应产物 HMBPP 与活性位点簇的结合。
