Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
J Am Chem Soc. 2022 Jun 29;144(25):11110-11119. doi: 10.1021/jacs.1c13580. Epub 2022 Jun 15.
Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from ( IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in DO, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.
肠道微生物对氨基酸衍生芳基乙酸的脱羧作用是一种具有挑战性的化学酶转化，会生成影响宿主生理的小分子。甘氨酰自由基酶（GRE）吲哚乙酸脱羧酶来自 （IAD）对吲哚-3-乙酸（I3A）进行非氧化自由基脱羧生成粪臭素，粪臭素是猪和反刍动物肠道中产生的一种与疾病相关的代谢物。尽管 IAD 很重要，但我们对其机制的了解有限。在这里，我们描述了 IAD 的机制，评估了先前提出的两个假设：（1）涉及 I3A 羧酸盐初始 1-e 氧化的 Kolbe 型脱羧反应，或（2）从 I3A 的α-碳原子上提取氢原子以生成初始碳中心自由基。定点突变、动力学同位素效应实验、在 DO 中进行的反应分析和计算建模与涉及初始氢原子转移的机制一致。这一发现扩展了 GRE 脱羧酶和非氧化脱羧酶采用的自由基机制的类型，更广泛地说。阐明 IAD 脱羧反应的机制可以增强我们对自由基酶的理解，并为调节这种与疾病相关的代谢提供信息。
