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NAD + ‐dependent DNA ligase ( Rv3014c ) from Mycobacterium tuberculosis : Novel structure‐function relationship and identification of a specific inhibitor
Author(s) -
Srivastava Sandeep Kumar,
Dube Divya,
Kukshal Vandana,
Jha Ashok Kumar,
Hajela Kanchan,
Ramachandran Ravishankar
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21457
Subject(s) - dna ligase , biology , dna ligases , nad+ kinase , liga , biochemistry , enzyme , mutant , in silico , microbiology and biotechnology , gene , medicine , alternative medicine , pathology , fabrication
Abstract Mycobacterium tuberculosis codes for an essential NAD + ‐dependent DNA ligase ( Mtu LigA) which is a novel, validated, and attractive drug target. We created mutants of the enzyme by systematically deleting domains from the C‐terminal end of the enzyme to probe for their functional roles in the DNA nick joining reaction. Deletion of just the BRCT domain from Mtu LigA resulted in total loss of activity in in vitro assays. However, the mutant could form an AMP‐ligase intermediate that suggests that the defects caused by deletion of the BRCT domain occur primarily at steps after enzyme adenylation. Furthermore, genetic complementation experiments using a LigA deficient E. coli strain demonstrates that the BRCT domain of Mtu LigA is necessary for bacterial survival in contrast to E. coli and T. filiformis LigA, respectively. We also report the identification, through virtual screening, of a novel N‐substituted tetracyclic indole that competes with NAD + and inhibits the enzyme with IC 50 in the low μ M range. It exhibits ∼15‐fold better affinity for Mtu LigA compared to human DNA ligase I. In vivo assays using LigA deficient S. typhimurium and E. coli strains suggest that the observed antibacterial activity of the inhibitor arises from specific inhibition of LigA over ATP ligases in the bacteria. In silico ligand‐docking studies suggest that the exquisite specificity of the inhibitor arises on account of its mimicking the interactions of NAD + with Mtu LigA. An analysis of conserved water in the binding site of the enzyme suggests strategies for synthesis of improved inhibitors with better specificity and potency. Proteins 2007. © 2007 Wiley‐Liss, Inc.