Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.
Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.
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理解并规避对天然硫酯底物对α-氧胺合酶反应的需求。
DOI：
10.1021/acschembio.2c00365
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发表时间：
2022-09-16
影响因子：
4
通讯作者：
Narayan, Alison R. H.
中科院分区：
文献类型：
--
作者：
Ackenhusen, Sarah E.;Wang, Ye;Chun, Stephanie W.;Narayan, Alison R. H.
许多酶类需要硫酯亲电体，如酰基载体蛋白和酰基辅酶A底物。对于体外应用，这些底物可能使这些化学转化不切实际。为了解决这一挑战，我们研究了辅酶A在α-氧代胺合酶SxtA AOS的门控催化中的机制。通过研究SxtA AOS和相应的酶变体对一组底物和辅酶A模拟物的反应性，我们确定活性是通过泛酰巯基乙胺臂和磷酸基团的结合来门控的，所述磷酸基团与残基Lys 154氢键结合，所述残基Lys 154通过AlphaFold 2模型预测位于通向活性位点的隧道中。为了提供一种经济的解决方案，用于生产规模的反应，原位转硫酯与泛酰巯基乙胺和简单的硫酯底物前体一起使用，导致生产性反应。这些研究结果概述了一种策略，采用ACP和CoA依赖性酶，是无法通过其他手段，而不需要成本高昂的辅酶A或载体蛋白活化底物。
Many enzyme classes require thioester electrophiles such as acyl-carrier proteins and acyl-coenzyme A substrates. For in vitro 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, in situ 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.
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影响因子：
15
作者：
Chun SW;Hinze ME;Skiba MA;Narayan ARH
通讯作者：
Narayan ARH
影响因子：
15
作者：
Chen, Mengbin;Liu, Chun-Ting;Tang, Yi
通讯作者：
Tang, Yi
影响因子：
3.9
作者：
Chalyk, Bohdan A.;Kandaurova, Inna Y.;Mykhailiuk, Pavel K.
通讯作者：
Mykhailiuk, Pavel K.
影响因子：
4.9
作者：
Heine, Daniel;Sundaram, Srividhya;Hertweck, Christian
通讯作者：
Hertweck, Christian
DOI：
10.1073/pnas.1918759117
发表时间：
2020-04-21
影响因子：
11.1
作者：
Dunbar, Kyle L.;Dell, Maria;Hertweck, Christian
通讯作者：
Hertweck, Christian