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. 2010 Dec 10;1(11):1011–1022. doi: 10.1007/s13238-010-0132-9

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

Chandrakala Basavannacharya 1, Paul R Moody 1, Tulika Munshi 1, Nora Cronin 1, Nicholas H Keep 1, Sanjib Bhakta 1,✉
PMCID: PMC3962982  EMSID: EMS57318  PMID: 21153518

Abstract

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 Nterminal residues, the other mutated residues are essential for catalysis. Variations in Km values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.

Keywords: peptidoglycan, MurE ligase, site-directed mutagenesis, m-DAP

References

  1. Barreteau H., Kovac A., Boniface A., Sova M., Gobec S., Blanot D. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008;32:168–207. doi: 10.1111/j.1574-6976.2008.00104.x. [DOI] [PubMed] [Google Scholar]
  2. Basavannacharya C., Robertson G., Munshi T., Keep N.H., Bhakta S. ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation. Tuberculosis (Edinb) 2010;90:16–24. doi: 10.1016/j.tube.2009.10.007. [DOI] [PubMed] [Google Scholar]
  3. Bertrand J.A., Auger G., Fanchon E., Martin L., Blanot D., van Heijenoort J., Dideberg O. Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli. EMBO J. 1997;16:3416–3425. doi: 10.1093/emboj/16.12.3416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertrand J.A., Auger G., Martin L., Fanchon E., Blanot D., Le Beller D., van Heijenoort J., Dideberg O. Determination of the MurD mechanism through crystallographic analysis of enzyme complexes. J Mol Biol. 1999;289:579–590. doi: 10.1006/jmbi.1999.2800. [DOI] [PubMed] [Google Scholar]
  5. Bognar A.L., Osborne C., Shane B. Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J Biol Chem. 1987;262:12337–12343. [PubMed] [Google Scholar]
  6. Boniface A., Bouhss A., Mengin-Lecreulx D., Blanot D. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity. J Biol Chem. 2006;281:15680–15686. doi: 10.1074/jbc.M506311200. [DOI] [PubMed] [Google Scholar]
  7. Bouhss A., Dementin S., Parquet C., Mengin-Lecreulx D., Bertrand J.A., Le Beller D., Dideberg O., van Heijenoort J., Blanot D. Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine:Dglutamate ligase (MurD) Biochemistry. 1999;38:12240–12247. doi: 10.1021/bi990517r. [DOI] [PubMed] [Google Scholar]
  8. Bouhss A., Mengin-Lecreulx D., Blanot D., van Heijenoort J., Parquet C. Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:Lalanine ligase from Escherichia coli. Biochemistry. 1997;36:11556–11563. doi: 10.1021/bi970797f. [DOI] [PubMed] [Google Scholar]
  9. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  10. Consaul S.A., Wright L.F., Mahapatra S., Crick D.C., Pavelka M.S., Jr. An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis. J Bacteriol. 2005;187:1612–1620. doi: 10.1128/JB.187.5.1612-1620.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dementin S., Bouhss A., Auger G., Parquet C., Mengin-Lecreulx D., Dideberg O., van Heijenoort J., Blanot D. Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. Eur J Biochem. 2001;268:5800–5807. doi: 10.1046/j.0014-2956.2001.02524.x. [DOI] [PubMed] [Google Scholar]
  12. Eveland S.S., Pompliano D.L., Anderson M.S. Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily. Biochemistry. 1997;36:6223–6229. doi: 10.1021/bi9701078. [DOI] [PubMed] [Google Scholar]
  13. Glaser P., Munier H., Gilles A.M., Krin E., Porumb T., Bârzu O., Sarfati R., Pellecuer C., Danchin A. Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis. EMBO J. 1991;10:1683–1688. doi: 10.1002/j.1460-2075.1991.tb07692.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glauner B., Höltje J.V., Schwarz U. The composition of the murein of Escherichia coli. J Biol Chem. 1988;263:10088–10095. [PubMed] [Google Scholar]
  15. Gordon E., Flouret B., Chantalat L., van Heijenoort J., Mengin-Lecreulx D., Dideberg O. Crystal structure of UDP-Nacetylmuramoyl-L-alanyl-D-glutamate: meso-diaminopimelate ligase from Escherichia coli. J Biol Chem. 2001;276:10999–11006. doi: 10.1074/jbc.M009835200. [DOI] [PubMed] [Google Scholar]
  16. Ikeda M., Wachi M., Jung H.K., Ishino F., Matsuhashi M. Nucleotide sequence involving murG and murC in the mra gene cluster region of Escherichia coli. Nucleic Acids Res. 1990;18:4014. doi: 10.1093/nar/18.13.4014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inoue M., Hamada S., Ooshima T., Kotani S., Kato K. Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme. Microbiol Immunol. 1979;23:319–328. doi: 10.1111/j.1348-0421.1979.tb00469.x. [DOI] [PubMed] [Google Scholar]
  18. Kawamoto I., Oka T., Nara T. Cell wall composition of Micromonospora olivoasterospora, Micromonospora sagamiensis, and related organisms. J Bacteriol. 1981;146:527–534. doi: 10.1128/jb.146.2.527-534.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Mahapatra S., Scherman H., Brennan P.J., Crick D.C. N Glycolylation of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium spp. is altered by drug treatment. J Bacteriol. 2005;187:2341–2347. doi: 10.1128/JB.187.7.2341-2347.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mengin-Lecreulx D., Blanot D., van Heijenoort J. Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli. J Bacteriol. 1994;176:4321–4327. doi: 10.1128/jb.176.14.4321-4327.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchell C., Oliver D. Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase. Mol Microbiol. 1993;10:483–497. doi: 10.1111/j.1365-2958.1993.tb00921.x. [DOI] [PubMed] [Google Scholar]
  23. Nanninga N. Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol. 1991;5:791–795. doi: 10.1111/j.1365-2958.1991.tb00751.x. [DOI] [PubMed] [Google Scholar]
  24. Sambrook J., Russell D.W. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001. [Google Scholar]
  25. Schleifer K.H., Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972;36:407–477. doi: 10.1128/br.36.4.407-477.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Story R.M., Steitz T.A. Structure of the recA protein-ADP complex. Nature. 1992;355:374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
  27. van der Wolk J.P., Klose M., de Wit J.G., den Blaauwen T., Freudl R., Driessen A.J. Identification of the magnesium-binding domain of the high-affinity ATP-binding site of the Bacillus subtilis and Escherichia coli SecA protein. J Biol Chem. 1995;270:18975–18982. doi: 10.1074/jbc.270.32.18975. [DOI] [PubMed] [Google Scholar]
  28. Vasstrand E., Jensen H.B., Miron T. Microbore single-column analysis of amino acids and amino sugars specific to bacterial cell wall peptidoglycans. Anal Biochem. 1980;105:154–158. doi: 10.1016/0003-2697(80)90438-8. [DOI] [PubMed] [Google Scholar]
  29. Velayati A.A., Masjedi M.R., Farnia P., Tabarsi P., Ghanavi J., Ziazarifi A.H., Hoffner S.E. Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. Chest. 2009;136:420–425. doi: 10.1378/chest.08-2427. [DOI] [PubMed] [Google Scholar]
  30. Walker J.E., Saraste M., Runswick M.J., Gay N.J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1:945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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