The Biosynthesis of Capuramycin-Type Antibiotics: Identification of the A-102395 Biosynthetic Gene Cluster, Mechanism of Self-Resistance, and Formation of Uridine-5′-Carboxamide [Microbiology]
April 8th, 2015 by Cai, W., Goswami, A., Yang, Z., Liu, X., Green, K. D., Barnard-Britson, S., Baba, S., Funabashi, M., Nonaka, K., Sunkara, M., Morris, A. J., Spork, A. P., Ducho, C., Garneau-Tsodikova, S., Thorson, J. S., Van Lanen, S. G.
A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent alpha-ketoglutarate:UMP dioxygenase and an L-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and L-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish L-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed permitting the inactivation of several genes including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395 thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.