Enzymatic Thioamide Formation in a Bacterial Antimetabolite Pathway

Abstract

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.

Bacterial pathogens: threat or treat (a review on bioactive natural products from bacterial pathogens)

“…20 and Table S1 †). [334][335][336][337] Thioguanine 51 is a sulfur-containing guanine analogue that works by disrupting DNA and RNA. Originally known as a synthetic compound, 333 thioguanine was rst isolated from the cultures of Pseudomonas sp.…”

“…337,339 The ATP-dependent YcfA enzyme catalyses the transfer of sulfur onto the guanine backbone 339 and uses a pyridoxal phosphate (PLP)-dependent specialised sulfur shuttle enzyme, YcfC that functions independently from the general sulfur mobilization pathways. 337 While the sulfur source in universal RNA-systems oen originates from L-cysteine through the action of cysteine desulfurases (IscS), 340 the cysteine-derived sulfur nucleophile in thioguanine biosynthesis is provided by YcfC and then, transferred and bound onto one of the cysteine residues (Cys113) of the YcfA active site. 337,339 Meanwhile, no thionated products were detected using the IscS homologue (Ea-IscS)-catalysed reaction in E. amylovora.…”

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa‐Perturbed Nucleotides

“…The pKa-perturbed 5FU is known to form stable Ag + -mediated base pairs, [24] especially at high pH, and to form Hg 2+ -5FU bonds under acidic conditions albeit of lower strength than the parent canonical thymidine. [20] On the other hand, the RNA-and DNA-targeting cytotoxin 6thioguanine [25] is capable of forming stable Cd 2+ -mediated base pairs. [26] Also, S6G-TP appears to be a good substrate for DNA polymerases and has been used to generate long, fully modified oligonucleotides that are not accessible by chemical methods.…”

Recent Advances in Our Understanding of the Biosynthesis of Sulfur Modifications in tRNAs

“…Alternatively, an additional sulfur carrier protein(s) may mediate between the MnmA and thiotetronate biosynthesis machinery. The 6-thioguanosine (s 6 G) modification is a virulence factor in the plant pathogen Erwinia amylovorans , and two proteins are required for the formation of s 6 G both in vivo and in vitro ( Litomska et al, 2018 ). The first, YcfC, is distantly related to PLP-dependent transferases such as cysteine desulfurases and carbon-sulfur lyases.…”