Genomics Research Center, Academia Sinica, Taipei, Taiwan.
The Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
Protein Sci. 2020 Jul;29(7):1655-1666. doi: 10.1002/pro.3879. Epub 2020 May 26.
Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMN complex (I), the FMN -N5-aloxyl-C'α -C4α zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMN an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.
虽然可以通过光谱法对一些黄素辅助的酶反应中的反应性黄素-N5/C4α-氧化物中间体进行分析，但其确切的化学结构却难以直观呈现。结构系统生物学和稳定同位素标记技术被用来纠正这种刻板的观点。通过对琥珀酸单加氧酶（Hmo）或其突变体 Y128F（单加氧酶）晶体进行浸泡实验，确定了三种过渡态类似物，即α-酮酸…N5-FMN 复合物（I）、FMN-N5-烷氧基-C'α-C4α 两性离子（II）和 FMN-N5-乙稀醇-N5-C4α-环氧化物（III），从而证实了 FMN 的 N5 可以作为替代反应中心在活性位点中极化生成类似叶立德的中间态。相比之下，从 Y128F 晶体浸泡在选定的底物中得到的四个不同的黄素-C4α-氧化物加合物（IV-VII），则证明了 FMN 的 C4α 是固有反应中心，见证了氧化、Baeyer-Villiger/过氧化物辅助脱羧和环氧化反应。结合停流动力学，首次在分子水平上对多方面的黄素依赖性反应连续体进行了物理剖析。
