Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Department of Biochemistry and Centre de recherche en biologie structurale, McGill University, Montréal, Canada H3G 0B1.
J Am Chem Soc. 2022 Aug 10;144(31):14057-14070. doi: 10.1021/jacs.1c13404. Epub 2022 Jul 27.
Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (C). We also obtain the crystal structure of a C domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-C modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-C identify several key residues for activity, including four residues that are mostly conserved in the C subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for C domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of C domains. Understanding C domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.
去氢氨基酸是天然产物的重要结构基元和生物合成中间体。许多非核糖体来源的生物活性天然产物含有去氢氨基酸；然而，大多数非核糖体肽中的去氢氨基酸生物合成还不太清楚。在这里，我们提供生化和生物信息学证据支持在脱水（C）中独特的一类缩合结构域的作用。我们还获得了参与抗生素甲氧基乙烯基甘氨酸生物合成的非核糖体肽合成酶 AmbE 的 C 结构域的晶体结构。生化分析表明，AmbE-C 修饰与供体载体蛋白相连的肽底物。AmbE-C 的突变研究确定了几个关键的活性残基，包括在 C 亚家族中大多数保守的四个残基。这些保守残基的丙氨酸突变要么显著增加要么降低 AmbE 活性。AmbE 表现出二聚体构象，这是不常见的，并且可以在不同的前体之间转移中间产物。我们的发现突出了 C 结构域在不同非核糖体肽途径中的统一去氢氨基酸生物合成中的核心脱水功能。我们的工作也开始揭示 C 结构域的机制。了解 C 结构域的功能可以促进含有去氢氨基酸的新型天然产物的鉴定，并能够将去氢氨基酸工程化到非核糖体肽中。
