ACS Chem Biol. 2022 Sep 16;17(9):2389-2395. doi: 10.1021/acschembio.2c00365. Epub 2022 Aug 16.
Many enzyme classes require thioester electrophiles such as acyl-carrier proteins and acyl-coenzyme A substrates. For applications, these substrates can render these chemical transformations impractical. To address this challenge, we have investigated the mechanism of coenzyme A in gating catalysis of one α-oxoamine synthase, SxtA AOS. Through investigating the reactivity of SxtA AOS and corresponding enzyme variants against a panel of substrates and coenzyme A mimics, we determined that activity is gated through the binding of the pantetheine arm and a phosphate group that hydrogen bonds to residue Lys154 that is predicted by an AlphaFold2 model to be located in a tunnel leading to the active site. To provide an economical solution for preparative-scale reactions, transthioesterification was used with pantetheine and simple thioester substrate precursors, resulting in productive reactions. These findings outline a strategy for employing ACP- and CoA-dependent enzymes that are inaccessible through other means without the need for cost-prohibitive coenzyme A or carrier protein-activated substrates.
许多酶类需要硫酯亲电试剂，如酰基载体蛋白和酰基辅酶 A 底物。对于应用来说，这些底物可能会使这些化学转化变得不切实际。为了应对这一挑战，我们研究了辅酶 A 在一种α-氧代胺合酶 SxtA AOS 催化中的门控作用机制。通过研究 SxtA AOS 及相应酶变体对一系列底物和辅酶 A 类似物的反应性，我们确定活性是通过 pantetheine 臂和一个磷酸基团的结合来控制的，该磷酸基团与 Lys154 残基形成氢键，Lys154 残基由 AlphaFold2 模型预测位于通向活性位点的隧道中。为了为制备规模的反应提供经济的解决方案，我们使用 pantetheine 和简单的硫酯底物前体进行转硫酯反应，从而得到了高产率的反应。这些发现为使用 ACP 和 CoA 依赖性酶提供了一种策略，这些酶通过其他方法无法获得，而不需要昂贵的辅酶 A 或载体蛋白激活的底物。
