Reactivity-Based Screening for Citrulline-Containing Natural Products Reveals a Family of Bacterial Peptidyl Arginine Deiminases

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is processed by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminal sequence threaded through the macrocycle. This unique lariat topology, which typically provides considerable stability toward heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of Arg deimination to yield citrulline (Cit). Although a Cit-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for Arg deimination has remained unknown. Herein, we describe the use of reactivity-based screening to discriminate bacterial strains that produce Arg- versus Cit-bearing citrulassins, yielding 13 new lasso peptide variants. Partial phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the Cit-containing variants. Absence of this gene correlated strongly with lasso peptide variants only containing Arg (i.e., des-citrulassin). Heterologous expression of the PAD gene in a des-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in Arg deimination. The PADs were then bioinformatically surveyed to provide a deeper understanding of their taxonomic distribution and genomic contexts and to facilitate future studies that will evaluate any additional biochemical roles for the superfamily.

核糖体合成且经翻译后修饰的肽（Ribosomally synthesized and post-translationally modified peptides, RiPPs）是一类天然产物家族，其定义特征为存在基因编码的前体肽，该前体肽由相关的生物合成酶加工后形成成熟产物。套索肽（lasso peptides）是RiPPs的一个子类，其标志性特征为N端氨基与内部天冬氨酸/谷氨酸残基之间形成异肽键，且C端序列穿过该大环结构。这种独特的套索拓扑结构通常对热与蛋白酶具有出色的稳定性，因此引发了学界将套索肽作为潜在治疗药物的研究兴趣。除了这类标志性的穿环大环内酰胺修饰外，已有报道称套索肽存在其他翻译后修饰，包括一例精氨酸脱亚胺生成瓜氨酸（citrulline, Cit）的修饰案例。尽管含瓜氨酸的套索肽（即瓜氨素，citrulassin）是在基因组导向的研究中偶然发现的，但负责精氨酸脱亚胺的基因始终未被探明。本文介绍了基于反应性的筛选方法，用于区分产生携带精氨酸与瓜氨酸的瓜氨素的细菌菌株，最终获得13种全新的套索肽变体。系统发育谱初步分析发现，一类远端编码的肽酰精氨酸脱亚胺酶（peptidyl arginine deiminase, PAD）基因广泛存在于含瓜氨酸的变体中；而该基因的缺失则与仅含精氨酸的套索肽变体（即脱瓜氨素，des-citrulassin）高度相关。将PAD基因在脱瓜氨素的产生菌株中进行异源表达后，成功检测到脱亚胺修饰的类似物，证实了PAD确实参与精氨酸脱亚胺过程。随后研究团队对PAD家族开展了生物信息学调研，以深入解析其分类学分布与基因组上下文，并为未来探究该超家族的其他生化功能提供便利。