A secondary kinetic isotope effect study of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement.
A secondary kinetic isotope effect study of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement.
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DOI：
10.1021/ja807987h
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发表时间：
2009-02-18
影响因子：
15
通讯作者：
Liu HW
中科院分区：
文献类型：
--
作者：
Munos JW;Pu X;Mansoorabadi SO;Kim HJ;Liu HW
1-脱氧-d-木酮糖 5-磷酸 (DXP) 还原异构酶（DXR，也称为甲基-d-赤藓糖醇 4-磷酸 (MEP) 合酶）是一种 NADPH 依赖性酶，在异戊二烯生物合成的非甲羟戊酸途径中催化 DXP 转化为 MEP。对于 DXR 催化反应，提出了两种机制。在 α-酮醇重排机制中，反应从 C-3 羟基的去质子化开始，然后进行 1,2-迁移，得到磷酸甲基赤藓糖，然后通过 NADPH 还原为 MEP。在逆羟醛/羟醛重排机制中，DXR首先以逆羟醛方式裂解DXP的C3-C4键，生成三碳和二碳磷酸双分子中间体。然后这两种物质通过羟醛反应重新结合，形成新的 C-C 键，产生醛中间体。随后 NADPH 的还原提供了 MEP。为了区分这些机制，我们制备了[3-2H]-和[4-2H]-DXP，并对DXR反应进行了竞争性二次动力学同位素效应（KIE）研究。 [3-2H]-和[4-2H]-DXP 观察到的正常 2° KIE 提供了令人信服的证据，支持 DXR 催化重排的逆羟醛/羟醛机制，其中限速步骤是 DXP C3-C4 键的裂解。
1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-d-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the non-mevalonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the α-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldol rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-2H]- and [4-2H]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2° KIEs observed for [3-2H]- and [4-2H]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.
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影响因子：
2.7
作者：
Phaosiri, C;Proteau, PJ
通讯作者：
Proteau, PJ
影响因子：
3.6
作者：
Fox, DT;Poulter, CD
通讯作者：
Poulter, CD
DOI：
10.1016/s0167-4838(96)00118-5
发表时间：
1996-11-14
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
影响因子：
--
作者：
Sculley, MJ;Morrison, JF;Cleland, WW
通讯作者：
Cleland, WW
DOI：
10.1073/pnas.95.17.9879
发表时间：
1998-08-18
影响因子：
11.1
作者：
Takahashi, S;Kuzuyama, T;Seto, H
通讯作者：
Seto, H
影响因子：
15
作者：
Thulasiram, Hirekodathakallu V.;Erickson, Hans K.;Poulter, C. Dale
通讯作者：
Poulter, C. Dale