Sulfur delivery mechanism in the biosynthesis of 6-thioguanine’s thioamide functional group
Embargo Date
2025-02-28
OA Version
Citation
Abstract
Sulfur is an essential element for living organisms that plays a crucial role in the activities of many natural products involved in both primary and secondary metabolism. Thio-nucleotides are a class of sulfur containing natural products mostly found in the anti-codon loop of tRNAs. Among them, 6-Thioguanine (6-TG) is unique, in that it is incorporated directly into the DNA where it can lead to cell death through regulation of the DNA mismatch repair mechanism. Recently, 6-TG was found to be a key virulence factor in Erwinia amylovora’s pathogenesis and its biosynthetic cluster was discovered. Two enzymes, YcfA, an AANH-like S-transferase, and YcfC, a PLP-dependent desulfurase, catalyze the sulfur insertion step. Initial characterization of the YcfA-YcfC reaction suggest that it does not require an exogenous reductant, unlike its homologous nucleotide S-transferases. However, the details of this novel sulfur insertion mechanism remain to be elucidated. Herein, the product of cysteine desulfuration by the YcfC protein is characterized, the YcfA and YcfC kinetics are explored, the cystine β-lyase activity of YcfC is elucidated, and peptide mass spectrometry evidence suggest that cysteine residues in YcfC can catalytically reduce the covalently linked YcfA-product complex. Finally, a new mechanistic model for the YcfA-YcfC catalyzed formation of 6-thioguanosine mono-phosphate from cysteine and guanosine monophosphate, in an adenosine tri-phosphate and pyridoxal-5-phosphate dependent manner is proposed.