Sulfur plays a critical role for the development and maintenance of life on earth, which is reflected by the wealth of primary metabolites, macromolecules, and cofactors bearing this element. Whereas a large body of knowledge has existed for sulfur trafficking in primary metabolism, the secondary metabolism involving sulfur has long been neglected. Yet, diverse sulfur functionalities have a major impact on the biological activities of natural products. Recent research at the genetic, biochemical, and chemical levels has unearthed a broad range of enzymes, sulfur shuttles, and chemical mechanisms for generating carbon-sulfur bonds. This Review will give the first systematic overview on enzymes catalyzing the formation of organosulfur natural products.

Sulfur for fire: The molecular basis for the biosynthesis of the antimetabolite 6-thioguanine (6TG) was unveiled in Erwinia amylovora, the causative agent of fire blight. Bioinformatics, heterologous pathway reconstitution in E. coli, and mutational analyses indicate that the protein YcfA mediates guanine thionation in analogy to 2-thiouridylase. Assays in planta and in cell cultures reveal for the first time a crucial role of 6TG in fire blight pathogenesis.

Schwefel und Feuerbrand: Die molekularen Grundlagen der Biosynthese des Antimetaboliten 6‐Thioguanin (6TG) wurden in dem Feuerbrand verursachenden Bakterium Erwinia amylovora aufgedeckt. Bioinformatik, die Rekonstitution der Biosynthese in E. coli und Mutationsanalysen zeigen, dass das Protein YcfA Guanin in Analogie zur 2‐Thiouridylase thioniert. Assays in Pflanzen und Zellkulturen decken eine wichtige Rolle von 6TG in der Pathogenese auf.

6-Thioguanine (6TG) is a DNA-targeting therapeutic used in the treatment of various cancers. While 6TG was rationally designed as a proof of concept for antimetabolite therapy, it is also a rare thioamide-bearing bacterial natural product and critical virulence factor of Erwinia amylovorans, plant pathogens that cause fire blight. Through gene expression, biochemical assays, and mutational analyses, we identified a specialized bipartite enzyme system, consisting of an ATP-dependent sulfur transferase (YcfA) and a sulfur-mobilizing enzyme (YcfC), that is responsible for the peculiar oxygen-by-sulfur substitution found in the biosynthesis of 6TG. Mechanistic and phylogenetic studies revealed that YcfA-mediated 6TG biosynthesis evolved from ancient tRNA modifications that support translational fidelity. The successful in vitro reconstitution of 6TG thioamidation showed that YcfA employs a specialized sulfur shuttle that markedly differs from universal RNA-related systems. This study sheds light on underexplored enzymatic C-S bond formation in natural product biosynthesis.

Fire blight is a devastating disease of Rosaceae plants, such as apple and pear trees. It is characterized by necrosis of plant tissue, caused by the phytopathogenic bacterium Erwinia amylovora. The plant pathogen produces the well-known antimetabolite 6-thioguanine (6TG), which plays a key role in fire blight pathogenesis. Here we report that YcfR, a member of the LTTR family, is a major regulator of 6TG biosynthesis in E. amylovora. Inactivation of the regulator gene (ycfR) led to dramatically decreased 6TG production. Infection assays with apple plants (Malus domestica cultivar Holsteiner Cox) and cell cultures of Sorbus aucuparia (mountain ash, rowan) revealed abortive fire blight pathogenesis and reduced plant response (biphenyl and dibenzofuran phytoalexin production). In the presence of the ΔycfR mutant, apple trees were capable of activating the abscission machinery to remove infected tissue. In addition to unveiling the regulation of 6TG biosynthesis in a major plant pathogen, we demonstrate for the first time that this antimetabolite plays a pivotal role in dysregulating the plant response to infection.