6‐Thioguanine (6TG) is a clinically used antitumor agent that was rationally designed as a DNA‐targeting antimetabolite, but it also occurs naturally. 6TG is a critical virulence factor produced by Erwinia amylovorans, a notorious plant pathogen that causes fire blight of pome fruit trees. The biosynthesis of the rare thioamide metabolite involves an adenylating enzyme (YcfA) and a sulfur‐mobilizing enzyme (YcfC), but the mechanism of sulfur transfer and putative intermediates have remained elusive. Through dissection and in vitro reconstitution of the thionation process using diverse substrates, we uncover an intermediate, prodrug‐like thio‐conjugate and elucidate the precise enzyme functions. YcfA not only adenylates GMP but also transfers the mercapto group of l‐cysteine to the activated carbonyl. A designated C‐S lyase (YcfC) then cleaves the resulting S‐adduct to yield the thioamide. This pathway is distinct from canonical tRNA sulfur modifications and known enzymatic peptide thionations. By exploring a wide range of substrate surrogates, we exploited the tolerance of the enzyme pair to produce even a seleno analogue. This study provides valuable insight into a previously unexplored area of bacterial thioamide formation and lays the groundwork for synthetic biology approaches to produce thioamide antimetabolites.

中文翻译：

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硫代和硒酰胺的酶促前药样途径


6-硫鸟嘌呤 (6TG) 是一种临床使用的抗肿瘤药物，被合理设计为 DNA 靶向抗代谢物，但它也自然存在。 6TG 是解淀粉欧文氏菌产生的关键毒力因子，解淀粉欧文氏菌是一种臭名昭著的植物病原体，可引起仁果树火疫病。罕见的硫代酰胺代谢物的生物合成涉及腺苷酸化酶（YcfA）和硫动员酶（YcfC），但硫转移的机制和假定的中间体仍然难以捉摸。通过使用不同底物对硫化过程进行解剖和体外重建，我们发现了中间体、前药样硫代缀合物，并阐明了精确的酶功能。 YcfA不仅使GMP腺苷酸化，而且还将L-半胱氨酸的巯基转移到活性羰基上。然后指定的 C-S 裂解酶 (YcfC) 裂解所得的 S-加合物，产生硫代酰胺。该途径不同于典型的 tRNA 硫修饰和已知的酶促肽硫化作用。通过探索各种底物替代物，我们利用酶对的耐受性甚至产生了硒代类似物。这项研究为以前未探索的细菌硫代酰胺形成领域提供了宝贵的见解，并为合成生物学方法生产硫代酰胺抗代谢物奠定了基础。