Biosynthesis of nucleoside antibiotics targeting bacterial translocase

New antibiotics are needed, particularly those that can be considered as new chemical entities and have novel targets relative to the current, clinical armament of antibiotics. Highly modified nucleoside antibiotics that inhibit bacterial translocase I (TL1) involved in cell wall biosynthesis fit both these descriptions, and have excellent potential in part because they are (i) nanomolar inhibitors of TL1, (ii) inhibit a target that has been proven to be essential for the survival of most, if not all, bacteria, and (iii) have no apparent toxicity in mice. We have defined the biosynthetic mechanism leading to the core dissacharyl-nucleoside structure of promising nucleoside antibiotics including A-90289 from Streptomyces sp. SANK 60405, muraminomicin from Streptosporangium sp, and muraymycin from Streptomyces sp. LL-AA896 using a combined in vivo and in vitro approach. The results have revealed a multi-enzyme pathway highlighted by bifurcation from the primary building block UMP and convergence to form the core. This data was utilized to scan the wealth of genomic information to identify a new lead antibiotic, sphaerimicin, that shares the nucleoside core structure. We now will accomplish the following specific aims: i) to define the mechanism of 3-amino-3-carboxypropionyl group attachment to the core, which is hypothesized to occur via a new enzyme strategy catalyzed by a bifunctional pyridoxal phosphatedependent protein and ii) define the biosynthetic mechanism of divergence from the dissacharylnucleoside nucleoside core that leads to unique multi-hybrid molecules including a nonribosomally derived peptide for muraymicin, an acylated diazepanone ring for A-90289 and muraminomicin, and a highly unusual fused piperidine ring system in sphaerimicin that is further elaborated by a branched, highly reduced polyketide.