Enzymatic malonylation of natural glycosides provided a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A “one-pot” system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated 2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1’s preference for glucosides. Furthermore, a mutant CtMaT1 with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

中文翻译：

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管花肉苁蓉具有底物混杂性和催化区域特异性的丙二酰转移酶的分子表征和结构基础

天然糖苷的酶法丙二酰化为类药物丙二酰化糖苷的供应提供了一种有前景的替代方法。然而，植物丙二酰转移酶的催化潜力和结构基础还远未完全阐明。这项工作从中鉴定出一种新的丙二酰转移酶 CtMaT1，它对具有不同苷元的多种糖苷表现出前所未有的单和/或二丙二酰化活性。建立了 CtMaT1 和丙二酰辅酶 A 合成酶的“一锅”系统来生物合成九种新的丙二酰化葡萄糖苷。结构研究表明，CtMaT1 拥有足够宽敞的酰基受体口袋，能够容纳不同的糖苷。此外，它还通过强电规和氢相互作用来识别丙二酰辅酶A。QM/MM计算揭示了CtMaT1的H167介导的2反应机制，而动态模拟检测到葡萄糖-6-OH基团与H167之间形成了稳定的氢键，导致其具有较高的丙二酰化区域特异性。两种异构糖苷的计算能量分布突显了对糖苷底物的较低反应能垒，强调了 CtMaT1 对糖苷的偏好。此外，还获得了二丙二酰化活性显着增加的突变体CtMaT1。通过 MM/GBSA 结合自由能计算阐明了潜在的分子机制。这项研究从功能和蛋白质结构的角度显着增进了对植物酰基转移酶的理解，同时也为酶促丙二酸化在药理学中的应用提供了多功能工具。