Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors
Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors
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DOI：
10.1073/pnas.1115749108
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发表时间：
2011-12-20
影响因子：
11.1
通讯作者：
Hemmi, Hisashi
中科院分区：
文献类型：
--
作者：
Nagai, Takuya;Unno, Hideaki;Hemmi, Hisashi
2型异戊烯基二磷酸异构酶（IDI-2）催化类异戊二烯生物合成的两个基本构件异戊烯基二磷酸和二甲基烯丙基二磷酸的异构化，通过还原黄素单核苷酸辅因子的质子化/去质子化的不寻常催化的证据。通过X射线晶体学和紫外-可见光谱研究了基于不可逆机理的抑制剂3-亚甲基-4-戊烯-1-基二磷酸或3-环氧乙烷基-3-丁烯-1-基二磷酸与黄素辅因子之间形成的共价加合物。结合黄素抑制剂加合物的IDI-2的晶体结构和加合物的紫外-可见光谱表明，共价键形成在黄素的C4 a，而不是在N5，这是以前提出的。此外，IDI-2-底物复合物的高分辨率晶体结构和新突变体的动力学研究证实，只有黄素辅因子可以催化底物的质子化，并表明黄素的N5最有可能参与质子转移。这些数据提供了一种机制，其中还原的黄素辅因子作为一种通用的酸/碱催化剂，并有助于稳定由质子化形成的碳阳离子中间体的支持。
Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanism-based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.