Chen Tzu-Yu, Kim Wantae, Guo Mo, Yao Angela, Zhang Y Jessie, Chang Wei-Chen
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA.
ACS Catal. 2025 Jul 18;15(14):12005-12013. doi: 10.1021/acscatal.5c03282. Epub 2025 Jun 30.
Isonitrile functional group is abundant in natural products and associated with various biological properties. To date, two pathways are known for isonitrile group installation. One is catalyzed by mononuclear nonheme iron dependent isonitrilases in which the reaction mechanism involves consecutive desaturation and decarboxylation processes. The other pathway includes isonitrile synthases, however, how isonitrile synthases enable the condensation of a sugar phosphate and an aromatic amino acid to install isonitrile group remains understudied. In this study, we examine substrate analogs and quantify reaction products using isotopologues to establish the substrate requirements of two isonitrile synthases, WelI1 and PIsnA, providing insights into their spatial and substrate requirements. Under experimental conditions, isonitrile synthases can only enable single turnover, with product binding likely associated with this observation. To reveal the reaction mechanism, we solved substrate- and product-bound protein structures. Furthermore, using a sequential soaking approach, we successfully trapped a plausible intermediate in crystallo. Observation of the plausible C2-imine intermediate in the active site suggests the reaction starts from the imine installation between the carbonyl group of the sugar phosphate and the amine group of the amino acid. Altogether, these studies provide insights into substrate recognition, i.e., pentose phosphate and L-aromatic amino acid, and the reaction mechanism of isonitrile synthases.
异腈官能团在天然产物中大量存在，并具有多种生物学特性。迄今为止，已知有两条引入异腈基团的途径。一条途径由单核非血红素铁依赖性异腈酶催化，其反应机制涉及连续的去饱和化和脱羧过程。另一条途径包括异腈合酶，然而，异腈合酶如何使磷酸糖和芳香族氨基酸缩合以引入异腈基团仍有待深入研究。在本研究中，我们研究了底物类似物，并使用同位素异构体对反应产物进行定量，以确定两种异腈合酶WelI1和PIsnA的底物需求，从而深入了解它们的空间和底物需求。在实验条件下，异腈合酶只能实现单轮反应，产物结合可能与这一观察结果有关。为了揭示反应机制，我们解析了结合底物和产物的蛋白质结构。此外，我们采用连续浸泡法，成功地在晶体中捕获了一个可能的中间体。在活性位点观察到可能的C2-亚胺中间体表明，反应始于磷酸糖的羰基与氨基酸的胺基之间的亚胺形成。总之，这些研究为底物识别（即磷酸戊糖和L-芳香族氨基酸）以及异腈合酶的反应机制提供了深入见解。
