Abstract
Tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death in the world. One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), the etiologic agent of TB. The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. In this work, 3D structure model of Mtb-MurA enzyme has been developed for the first time by homology modeling and molecular dynamics simulation techniques. Multiple sequence alignment and 3D structure model provided the putative substrate binding pocket of Mtb-MurA with respect to E. coli MurA. This analysis was helpful in identifying the binding sites and molecular function of the MurA homologue. Molecular docking study was performed on this 3D structure model, using different classes of inhibitors like fosfomycin, cyclic disulfide analog RWJ-3981, pyrazolopyrimidine analog RWJ-110192, purine analog RWJ-140998, 5-sulfonoxy-anthranilic acid derivatives T6361, T6362 and the results showed that the 5-sulfonoxyanthranilic acid derivatives showed the best interaction compared to other inhibitors. We also designed new efficient analogs of T6361 and T6362 which showed even better interaction with Mtb-MurA than the parental 5-sulfonoxy-anthranilic acid derivatives. Further the comparative molecular electrostatic potential and cavity depth analysis of Mtb-MurA suggested several important differences in its substrate and inhibitor binding pocket. Such differences could be exploited in the future for designing a more specific inhibitor for Mtb-MurA enzyme.
Similar content being viewed by others
References
Altschul, S.F., Grish, W., Miller, W., Myers, E.W., Lipman, D.J. 1990. Basic local alignment search tool. J Mol Biol 215, 403–410.
Andersen, P. 2007. Tuberculosis vaccines — an update. Nat Rev Microbiol 5, 484–487.
Anuradha, C.M., Mulakayala, C., Babajan, B., Naveen, M., Rajasekhar, C. 2009. Probing ligand binding modes of Mycobacterium tuberculosis MurC ligase by molecular modeling, dynamics simulation and docking. J Mol Model 16, 77–85.
Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C.M., Harris, D.E., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E.II., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R.M., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L.D., Oliver, S., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter, S., Seeger, K., Skelton, S., Squares, S., Squares, R., Sulston, J.E., Taylor, K., Whitehead, S., Barrell, B.G. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.
Connolly, M.L. 1983. Solvent-accessible surfaces of proteins and nucleic acids. Science 221, 709–713.
Cox, H.S., Morrow, M., Deutschmann, P.W. 2008. Long term efficacy of DOTS regimens for tuberculosis systematic review. BMJ 336, 484–487.
de Smet, K.A., Kempsell, K.E., Gallagher, A., Duncan, K., Young, D.B. 1999. Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology 145, 3177–3184.
Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., Liang, J. 2006. CASTp: Computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acid Research 34, 116–118.
Ellen, Z.B., Deborah, A., Montenegro, S., Lisa, L., Ignatitus, T., Glenda, C.W., Barbard, D.F., Karen, B. 2001. Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrob Agents Che 45, 3182–3188.
Eschenburg, S., Priestman, M.A., Abdul-Latif, F.A., Delachaume, C., Fassy, F., Schonbrunn, E. 2005. A novel inhibitor that suspends the induced fit mechanism of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). J Biol Chem 280, 14070–14075.
Hess, B., Bekker, H., Berendsen, H., Fraaije, J. 1997. LINCS: A linear constraint solver for molecular simulations. J Comp Chem 18, 1463–1472.
Huey, R., Morris, G.M., Olson, A.J., Goodsell, D.S. 2007. A semiempirical free energy force field with charge-based desolvation. J Comp Chem 28, 145–152.
Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G., Gibson, T.J. 1998. Multiple sequence alignment with ClustalX. Trends in Biochem Sci 10, 403–405.
Kim, D.H., Lees, W.J., Kempsell, K.E., Lane, W.S., Duncan, K., Walsh, C.T. 1996. Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry 35, 4923–4928.
Laskowski, R.A., Chistyakov, V.V., Thornton, J.M. 2005. PDBSUM more: New summaries and analyses of the known 3-D structures proteins and nucleic acids. Nucleic Acids Res 33, 266–268.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M. 1993. PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283–291.
Marquardt, J.L., Siegele, D.A., Kolter, R., Walsh, C.T. 1992. Cloning and sequencing of Escherichia coli murZ and puri?cation of its product, a UDP-Nacetylglucosamine enolpyruvyl transferase. J Bacteriol 74, 5748–5752.
McCoy, A.J., Sandlin, R.C., Maurelli, A.T. 2003. In vitro and in vivo functional activity of chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J Bacteriol 185, 1218–1228.
Miyamoto, S., Kollman, P.A. 1992. SETTLE: An analytical version of the SHAKE and RATTLE algorithms for rigid water models. J Comp Chem 13, 952–962.
Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., Olson, A.J. 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comp Chem 19, 1639–1662.
Parrish, N.M., Kuhajda, F.P., Heine, H.S., Bishai, W.R., Dick, J.D. 1999. Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis. J Antimicrob Chemother 43, 219–226.
Philip, W.J., Poulet, S., Eiglmeier, K., Pascopella, Balasubramanian, V., Heym, B., Bergh, S., Bloom, B.R., Jacobs, W.R. Jr, Cole, S.T. 1996. An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv and comparison with Mycobacterium leprae. Proc Natl Acad Sci USA 93, 3132–3137.
Raetz, C.R., Roderick, S.L. 1995. A left-handed parallel beta helix in the structure of UDP-Nacetylglucosamine acyltransferase. Science 270, 997–1000.
Rocchia, W., Sridharan, S., Nicholls, A., Alexov, E., Chiabrera, A., Honig, B. 2002. Rapid grid-based construction of the molecular surface for both molecules and geometric objects: applications to the finite difference Poisson-Boltzmann method. J Comp Chem 23, 128–137.
Roy, E., Lowrie, D.B., Jolles, S.R. 2007. Current strategies in TB immunotherapy. Curr Mol Med 7, 373–386.
Sali, A., Blundell, T.L. 1993. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234, 779–815.
Sanner, M.F. 1999. Python A programming language for software integration and development. J Mol Graph Model 17, 57–60.
Schonbrunn, E., Sack, S., Eschenburg, S., Perrakis, A., Krekel, F., Amrhein, N., Mandelkow, E. 1996. Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin. Structure 4, 1065–1075.
Schuettelkopf, A.W., van Aalten, D.M.F. 2004. PRODRG a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallographica 60, 1355–1363.
Seth, V., Beotra, A., Semwal, O.P., Mukhopadhya, S. 1990. Monitoring of serum rifampin and isoniazid levels in childhood tuberculosis. Am Rev Respir Dis, 141, 330–337.
Sinha, N., Smith-Gill, S.J. 2002. Electrostatics in protein binding and function. Curr Protein Pept Sci 3, 601–614.
Sippl, M.J. 1993. Recognition of errors in three-dimensional structures in proteins. Proteins 17, 355–362.
Skarzynski, T., Kim, D.H., Lees, W.J., Walsh, C.T., Duncan, K. 1998. Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA. Biochemistry 37, 2572–2577.
Skarzynski, T., Mistry, A., Wonacott, A., Hutchinson, S.E., Kelly, V.A., Duncan, K. 1996. Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-Nacetylglucosamine and the drug fosfomycin. Structure 4, 1465–1474.
Snider, D.E., Raviglione, M., Kochi, A. 1993. Global burden of tuberculosis. In: Bloom, B. (Ed.) Tuberculosis: Pathogenesis, Protection and Control, 1st Edition, ASM Press, Washington, DC, 3.
Stallings, W.C., Abdel-Meguid, S.S., Lim, L.W., Shieh, H.S., Dayringer, H.E., Leimgruber, N.K., Stegeman, R.A., Anderson, K.S., Sikorski, J.A., Padgette, S.R., Kishore, G.M. 1991. Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: A distinctive protein fold. Proc Natl Acad Sci USA 88, 5046–5050.
van der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A.E., Berendsen, H.J. 2005. GROMACS: Fast, flexible, and free. J Comput Chem 26, 1701–1718.
Villarreal-Ramos, B. 2009. Towards improved understanding of protective mechanisms induced by the BCG vaccine. Expert Rev Vaccines 8, 1531–1534.
Vriend, G. 1990. WHAT IF: A molecular modeling and drug design program. J Mol Graph 8, 52–56.
Wang, R., Gao, Y., Lai, L. 2000. LigBuilder: A multipurpose program for structure-based drug design. J Mol Model 6, 498–516.
Weiner, P.K., Langridge, R., Blaney, J.M., Schaefer, R., Kollman, P.A. 1982. Electrostatic potential molecular surfaces. Proc Natl Acad Sci USA 79, 3754–3758.
Wright, A., Zignol, M., van Deun, A. 2009. Epidemiology of antituberculosis drug resistance 2002-07: An updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Lancet. 373, 1861–1873.
Zoeiby, E.L., Sanschagrin, F., Levesque, R.C. 2003. Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 47, 1–12.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Babajan, B., Chaitanya, M., Rajsekhar, C. et al. Comprehensive structural and functional characterization of Mycobacterium tuberculosis UDP-NAG enolpyruvyl transferase (Mtb-MurA) and prediction of its accurate binding affinities with inhibitors. Interdiscip Sci Comput Life Sci 3, 204–216 (2011). https://doi.org/10.1007/s12539-011-0100-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12539-011-0100-y