The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

中文翻译：

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α-羟酸氧化酶中的黄素单核苷酸辅因子在控制底物氧化水平方面发挥其亲电/亲核对偶性。

对羟基扁桃酸酯氧化酶（Hmo）的Y128F单个突变体能够通过四电子氧化脱羧反应将扁桃酸酯氧化为苯甲酸酯。当使用甲酰苯甲酸酯（第一次双电子氧化的产物）和过氧化氢（氧化剂）作为底物时，反应没有进行，这表明游离的过氧化氢不是第二次双电子氧化中的定型氧化剂。黄素单核苷酸（FMN）依赖的四电子氧化反应如何发生仍然是未知的。结构和生化探索为这个问题提供了新的思路。Hmo及其配体与或不与底物配体的15个高分辨率晶体结构揭示了氧化的FMN（FMNox）具有以前未知的亲电/亲核对偶性。在Y128F突变体中，活性位点扰动集合体促进FMNox极化为亲核叶立德，后者可以作用于α-酮酸，形成N5-酰基-FMNred死端加合物。在四电子氧化中，经由N5-链烷醇-FMNredC'α碳负离子中间体的分子内歧化反应可解释ThDP / PLP / NADPH独立的氧化脱羧反应。合成的5-脱氮-FMNox辅因子与α-羟酰胺或α-酮酰胺的结合在生化和结构上均支持所提出的机理。α碳负离子中间体可能是不依赖ThDP / PLP / NADPH的氧化脱羧反应的原因。合成的5-脱氮-FMNox辅因子与α-羟酰胺或α-酮酰胺的结合在生化和结构上均支持所提出的机理。α碳负离子中间体可能是不依赖ThDP / PLP / NADPH的氧化脱羧反应的原因。合成的5-脱氮-FMNox辅因子与α-羟酰胺或α-酮酰胺的结合在生化和结构上均支持所提出的机理。