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J. Biol. Chem., Vol. 280, Issue 34, 30273-30281, August 26, 2005

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

CRYSTAL STRUCTURE OF THE ADENYLATION DOMAIN AND IDENTIFICATION OF NOVEL INHIBITORS^*

Sandeep Kumar Srivastava, Rama Pati Tripathi¶, and Ravishankar Ramachandran||

From the Divisions Molecular and Structural Biology and ^¶Medicinal and Process Chemistry, Central Drug Research Institute, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India

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.

Received for publication, April 7, 2005 , and in revised form, May 10, 2005.

The atomic coordinates and structure factors (code 1ZAU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The work was supported in part by Department of Biotechnology Grant SSP0139 and Council for Scientific and Industrial Research Network Grant CMM0017. This is Communication 6757 from the Central Drug Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of junior and senior research fellowships from the Council for Scientific and Industrial Research.

^|| To whom correspondence should be addressed: Molecular and Structural Biology Div., Central Drug Research Institute, P. O. Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India. Tel.:91-522-261-2411 (ext. 4442); Fax: 91-522-262-3405; E-mail: ravi_anitha{at}yahoo.com.

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