NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis

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.DNA ligases are vital enzymes in replication and repair and catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA through similar mechanisms (1). These enzymes can be divided into two classes, viz. NAD ؉ -and ATP-dependent ligases, based on the cofactor specificities (2). NAD ؉ -dependent DNA ligases, commonly called LigA, are found in bacteria and entomopoxviruses (3, 20), whereas ATP-dependent ligases are ubiquitous (3). Although there is little sequence homology between the eubacterial and eukaryotic enzymes, they exhibit some structural homology in specific domains (4, 5). The mechanistic steps involved in enzymatic action are also broadly conserved. Briefly, in the first step, the mode of action involves an attack on the ␣-phosphorus of ATP or NAD ؉ by the enzyme, releasing pyrophosphate or NMN and forming a ligase-adenylate intermediate. In the second step, the bound AMP is transferred to the 5Ј-end of DNA to form a DNAadenylate intermediate. AMP is released in the third step, where the protein catalyzes the joining of the 3Ј-nicked DNA to the DNA-adenylate intermediate. These steps involve large conformational changes and also encircling and partial unwinding of the nicked DNA substrate (6 -8).Some bacteria code for both NAD ؉ -and ATP-dependent DNA ligases (3, 9). Mycobacterium tuberculosis codes for at least three different types of ATP-dependent ligases and a NAD ϩ -dependent ligase (10, 11). Gene knockout and other studies have shown LigA to be indispensable in several bacteria, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and M. tuberculosis (10,(12)(13)(14)(15).No LigA structure from mycobacterial sources is available to date. However, the crystal structure of the full-length protein is available for the Thermus filifo...

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA)

“…The 671-amino acid E. coli LigA protein consists of a core nucleotidyltransferase (NTase) 2 domain (amino acids 70 -316) flanked by an N-terminal Ia domain (amino acids 1-69) and four C-terminal modules: an OB-fold domain (amino acids 317-404), a tetracysteine zinc finger domain (amino acids 405-432), a helix-hairpin-helix domain (amino acids 433-586), and a BRCT domain (amino acids 587-671) (5)(6)(7)(8)(9). Each step of the ligation pathway depends on a different subset of the LigA domain modules, with only the NTase domain being required for all steps.…”

Synthesis and anticancer activity of novel 2-substituted pyranopyridine derivatives

“…In addition, 2-amino pyridine nucleus is widely used in the production of various drugs such as piroxicam and tenoxicam (non-steroidal anti-inflammatory and analgesic drugs), sulfapyridine (antibacterial drug) and tripelennamine (antipruritic and the first generation antihistaminic), and triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) has entered phase I and II clinical trials as an antitumor chemotherapeutic agent [15]. Furthermore, the fused bicyclic ring pyranopyridine constitutes an important class of heterocyclic compounds having diverse biological activities such as anti-allergic, anti-inflammatory, estrogenic [16,17], anti-proliferative [18], anti-bacterial (including antitubercular) [19], antimyopic [20], hypotensive [21], anti-rheumatic [22] and antiasthmatic [23]. On the other hand, over the past few years, some research groupshave been interested in introducing a comprehensive study program towards the synthesis of thiophenes and their fused derivatives [24][25][26][27].…”

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A novel series of 2-aminopyranopyridine derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) were synthesized utilizing 2-chloro-4-(thiophen-2-yl)-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile (2) as a key starting compound. The structures of the newly synthesized compounds were confirmed on the basis of elemental analyses and infrared (IR), 1 H, 13 C-nuclear magnetic resonance (NMR) and mass spectra.

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Fe3O4@SiO2–NH2core-shell nanocomposite as an efficient and green catalyst for the multi-component synthesis of highly substituted chromeno[2,3-b]pyridines in aqueous ethanol media

“…In addition, chromenopyridines have been reported to have antiproliferative (31), cancer chemopreventive (32), antibacterial (including antitubercular) (33), antimyopic (34), antihistaminic (35), hypotensive (36), antirheumatic (37), and antiasthmatic activities (38). The synthetic routes for the preparation of substituted chromenopyridines mainly involve: the reaction of 4-chlorocoumarin-3-carbaldehyde with malononitrile (39), 2-amino-3-formylchromone with cyclic active methylene compounds, 2-amino-4-(phenylsulfanyl)-4H-chromene-3-carbonitrile and malononitrile (40), 2,2-disubstituted chroman-4-one with aromatic aldehydes and 2-cyanoacetamide (41), 4-chloro-2H-3-chromene carbaldehydes on reaction with ethyl-3-aminocrotonate (42) and the reaction of 2,2-disubstituted chroman-4-one with aromatic aldehydes and 2-cyanoacetamide (43).…”