Plants contain rapidly evolving specialized metabolic enzymes to support the synthesis of a myriad of functionally diverse natural products. In the case of coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered in Arabidopsis that catalyzes coumarin formation from o-hydroxylated trans-hydroxycinnamoyl-CoA substrates. COSY is the first and only BAHD enzyme known to date that catalyzes an intramolecular acyl transfer reaction. Here we combine structural, biochemical, and computational approaches to investigate the mechanistic basis for the unique coumarin synthase activity of COSY. Comparative analyses of crystal structures of Arabidopsis thaliana COSY relative to other BAHD proteins reveal that COSY possesses an unconventional active-site configuration adapted to its specialized activity. Through deuterium exchange experiments, we discover a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Mutagenesis studies and quantum mechanical cluster modeling further support that this mechanism is key to COSY’s ability to lower the activation energy of the trans-to-cis isomerization of the hydroxycinnamoyl-CoA substrates, a critical rate-limiting step leading to coumarin production. This study unveils the emergence of an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on the potential evolutionary origin of COSY and its recruitment to the evolutionarily new coumarin biosynthetic pathway in eudicots.

中文翻译：

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植物香豆素合酶 COSY 中基于质子交换的异构化和内酯化机制的出现

植物含有快速进化的专门代谢酶，以支持无数功能多样的天然产物的合成。在香豆素生物合成的情况下，最近在拟南芥中发现了一种 BAHD 酰基转移酶家族酶 COSY，它催化邻羟基化反式羟基肉桂酰辅酶 A 底物形成香豆素。COSY 是迄今为止已知的第一种也是唯一一种催化分子内酰基转移反应的 BAHD 酶。在这里，我们结合结构、生物化学和计算方法来研究 COSY 独特的香豆素合酶活性的机制基础。拟南芥晶体结构比较分析与其他 BAHD 蛋白相关的 COSY 表明 COSY 拥有适应其专门活动的非常规活性位点配置。通过氘交换实验，我们在 COSY 的催化循环过程中发现了邻羟基化反式羟基肉桂酰辅酶 A 底物的 α-碳处独特的质子交换机制。诱变研究和量子力学簇模型进一步支持这种机制是COSY降低反式-顺式活化能的关键羟基肉桂酰辅酶 A 底物的异构化，这是导致香豆素产生的关键限速步骤。这项研究揭示了一种由 BAHD 家族酶介导的非常规催化机制的出现，并阐明了 COSY 的潜在进化起源及其在真双子叶植物中进化的新香豆素生物合成途径中的募集。