Executive Summary : | Antibiotics have played a critical role in fighting deadly infectious diseases. However, microbial resistance to the currently employed antibiotics is becoming a significant concern for human health worldwide. Hence, there is an urgent need to discover new antibiotics with novel structures and mechanisms. Natural products and their derivatives constitute one of the primary sources of antibiotics. With the advent of the genomic revolution, natural product-based drug discovery has been revitalized recently. Nucleosides, consisting of a nucleobase and a structurally diverse sugar unit, are an emerging class of natural products with promising therapeutic properties. They are classified as N- or C-nucleosides. Although clinically very useful, N-nucleosides suffer from poor stability due to a hydrolyzable C-N glycosidic linkage. Hence, there has been considerable interest in developing C-nucleosides with a stable C-C glycosidic bond as potential drug candidates. Pseudouridimycin (PUM) is a potent peptidyl C-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP), the essential enzyme responsible for RNA synthesis in bacteria (IC50 0.1 µM). It functions as a nucleoside-analog inhibitor of RNAP by blocking the binding of uridine triphosphate, thus disrupting RNA synthesis. PUM exhibits broad-spectrum antibiotic activity with impressive potency. Interestingly, PUM does not inhibit human RNAPs, thus providing selectivity for clinical use. This mode of action differs from the other therapeutically used allosteric inhibitors of RNAP (such as rifamycins). Since PUM targets a critical region of RNAP, the emergence of resistance is low, making it a potential lead for antibiotic development. Besides its novel mode of action, PUM also possesses a remarkable structure comprising a formamidinylated, N-hydroxylated dipeptide with a pseudouridine scaffold. Although pseudouridine is an abundant modification in RNA, bioactive metabolites with pseudouridine are rare. Recently, the biosynthetic gene cluster for PUM has been discovered in Streptomyces sp. ID38640 and a pathway has been proposed based on the preliminary studies (in-vivo genetic knockouts, bioinformatics, etc.). However, none of the enzymes involved in PUM biosynthesis have been biochemically characterized. This project is aimed at in-vitro reconstitution and mechanistic investigation of these intriguing enzymes (pseudouridine synthase, flavin-dependent oxidoreductase, N-hydroxylase, ATP-grasp ligases, PLP-dependent aminotransferase, etc.) using various biochemical/structural studies on the recombinant proteins. Besides unraveling the elegant organic chemistry of the enzymes in synthesizing the dipeptidyl C-nucleoside framework of PUM, this study will enable us to design combinatorial biosynthetic protocols to generate a library of PUM analogs. The findings will help in discovering new C-nucleosides via genome mining and in developing potent derivatives to combat antibiotic resistance in future. |