title: NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.
abstract: DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.
authors.affiliation: Division Molecular and Structural Biology, Central Drug Research Institute, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India.
authors.forename: Sandeep Kumar
authors.initials: SK
authors.lastname: Srivastava
authors.forename: Rama Pati
authors.initials: RP
authors.lastname: Tripathi
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors
chemical_list: D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D004247:DNA; D011088:DNA Ligases; C120640:DNA ligase (NAD)