Institute of Biochemistry and Food Chemistry, Food Chemistry Division, University of Hamburg, Grindelallee 117, D-20146 Hamburg, Germany.
J Am Chem Soc. 2010 Mar 10;132(9):2983-90. doi: 10.1021/ja908395r.
Riboflavin synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of riboflavin synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
核黄素合酶催化两个底物分子（6,7-二甲基-8-核糖基色胺）之间的四碳片段转移，形成核黄素和 5-氨基-6-核糖基氨基-2,4(1H,3H)-嘧啶二酮。早期研究表明，两个底物分子形成的五员环加合物是一种催化有效的中间体，但该中间体的形成机制仍不清楚。本研究表明，来自大肠杆菌的核黄素合酶的重组 N 端结构域与 6,7-二甲基-8-核糖基色胺的亚甲烯型阴离子特异性相互作用，但不与水溶液中占优势的三环加合物型阴离子中的任何一种相互作用。虽然这些发现可以应用于以前发表的机制假说中，但我们还提出了一个新的、假设的反应序列，该序列从 6,7-二甲基-8-核糖基色胺亚甲烯阴离子向电中性的 6,7-二甲基-8-核糖基色胺分子转移氢原子开始。由这种氢转移产生的一对脱水色胺和二氢色胺分子被提议进行 4 + 2 环加成，从而生成实验记录的五员环中间体。与需要亲核试剂参与的早期机制概念（该概念不支持结构和突变体数据）不同，新的概念没有这样的要求。此外，它需要更少的反应步骤，并且与所有实验数据一致。
