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Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.

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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,… Click to show full abstract

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.

Keywords: oxoamine synthase; understanding circumventing; native thioester; circumventing requirement; requirement native; thioester

Journal Title: ACS chemical biology
Year Published: 2022

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