The AAPS Journal

, 20:44 | Cite as

Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from Mycobacterium tuberculosis and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation

  • Kuppan Gokulan
  • Sangeeta Khare
  • Carl E. Cerniglia
  • Steven L. Foley
  • Kottayil I. Varughese
Research Article Theme: Better Drugs for Better Life: Drug Discovery and Development Colloquium 2017
Part of the following topical collections:
  1. Theme: Better Drugs for Better Life: Drug Discovery and Development Colloquium 2017


The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4➔3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3➔3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of β-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.


β-lactamase D,D-transpeptidase imipenem L,D-transpeptidase meropenem 



The authors thank Dr. Henry Bellamy, CAMD, for SAD data collection, and SSRL for high resolution data collection. Dr. Kuppan Gokulan is supported by the FDA Commissioner Fellowship Program. The authors would like to thank Dr. Kevin Young (UAMS), and Dr. Sutherland (NCTR) for their helpful review and critique of the manuscript.

Supplementary material

12248_2018_193_MOESM1_ESM.doc (778 kb)
ESM 1 (DOC 778 kb)
12248_2018_193_MOESM2_ESM.doc (12 kb)
ESM 2 (DOC 12.5 kb)


  1. 1.
    Rogers HJ, James CA, Morrison PJ, Bradbrook ID. Effect of cimetidine on oral absorption of ampicillin and cotrimoxazole. J Antimicrob Chemother. 1980;6(2):297–300. Scholar
  2. 2.
    van Heijenoort J. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology. 2001;11(3):25R–36R. Scholar
  3. 3.
    Ghuysen JM, Goffin C. Lack of cell wall peptidoglycan versus penicillin sensitivity: new insights into the chlamydial anomaly. Antimicrob Agents Chemother. 1999;43(10):2339–44.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Vollmer W, Holtje JV. The architecture of the murein (peptidoglycan) in gram-negative bacteria: vertical scaffold or horizontal layer(s)? J Bacteriol. 2004;186(18):5978–87. Scholar
  5. 5.
    Matsuhashi M. Biosynthesis in the bacterial cell wall. Tanpakushitsu Kakusan Koso. 1966;11(10):875–86.PubMedGoogle Scholar
  6. 6.
    Jamin M, Wilkin JM, Frere JM. Bacterial DD-transpeptidases and penicillin. Essays Biochem. 1995;29:1–24.PubMedGoogle Scholar
  7. 7.
    Goffin C, Ghuysen JM. Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent. Microbiol Mol Biol Rev. 2002;66(4):702–38, table of contents. Scholar
  8. 8.
    Lavollay M, Arthur M, Fourgeaud M, Dubost L, Marie A, Riegel P, et al. The beta-lactam-sensitive D,D-carboxypeptidase activity of Pbp4 controls the L,D and D,D transpeptidation pathways in Corynebacterium jeikeium. Mol Microbiol. 2009;74(3):650–61. CrossRefPubMedGoogle Scholar
  9. 9.
    Schleifer KH, Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972;36(4):407–77.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Mainardi JL, Legrand R, Arthur M, Schoot B, van Heijenoort J, Gutmann L. Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium. J Biol Chem. 2000;275(22):16490–6. CrossRefPubMedGoogle Scholar
  11. 11.
    Gupta R, Lavollay M, Mainardi JL, Arthur M, Bishai WR, Lamichhane G. The mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat Med. 2010;16(4):466–9. Scholar
  12. 12.
    Peltier J, Courtin P, El Meouche I, Lemee L, Chapot-Chartier MP, Pons JL. Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links. J Biol Chem. 2011;286(33):29053–62. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kuzin AP, Liu H, Kelly JA, Knox JR. Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases. Biochemistry. 1995;34(29):9532–40. Scholar
  14. 14.
    Bugg TD, Braddick D, Dowson CG, Roper DI. Bacterial cell wall assembly: still an attractive antibacterial target. Trends Biotechnol. 2011;29(4):167–73. Scholar
  15. 15.
    Hugonnet JE, Tremblay LW, Boshoff HI, Barry CE 3rd, Blanchard JS. Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science. 2009;323(5918):1215–8. Scholar
  16. 16.
    Erdemli SB, Gupta R, Bishai WR, Lamichhane G, Amzel LM, Bianchet MA. Targeting the cell wall of Mycobacterium tuberculosis: structure and mechanism of L,D-transpeptidase 2. Structure. 2012;20(12):2103–15. Scholar
  17. 17.
    Kim HS, Kim J, Im HN, Yoon JY, An DR, Yoon HJ, et al. Structural basis for the inhibition of Mycobacterium tuberculosis L,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 3):420–31. Scholar
  18. 18.
    Both D, Steiner EM, Stadler D, Lindqvist Y, Schnell R, Schneider G. Structure of LdtMt2, an L,D-transpeptidase from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 3):432–41. Scholar
  19. 19.
    Correale S, Esposito C, Pirone L, Vitagliano L, Di Gaetano S, Pedone E. A biophysical characterization of the folded domains of KCTD12: insights into interaction with the GABAB2 receptor. J Mol Recognit. 2013;26(10):488–95. Scholar
  20. 20.
    Lecoq L, Bougault C, Triboulet S, Dubee V, Hugonnet JE, Arthur M, et al. Chemical shift perturbations induced by the acylation of Enterococcus faecium L,D-transpeptidase catalytic cysteine with ertapenem. Biomol NMR Assign. 2014;8(2):339–43. Scholar
  21. 21.
    Li WJLD, Hu YL, Zhang XE, Bi LJ, Wang DC. Crystal structure of L,D-transpeptidase LdtMt2 in complex with meropenem reveals the mechanism of carbapenem against Mycobacterium tuberculosis. Cell Res. 2013;23(5):728–31. Scholar
  22. 22.
    Van Duyne GD, Standaert RF, Karplus PA, Schreiber SL, Clardy J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J Mol Biol. 1993;229(1):105–24. Scholar
  23. 23.
    Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–26. Scholar
  24. 24.
    Cruickshank DW. Remarks about protein structure precision. Acta Crystallogr D Biol Crystallogr. 1999;55(3):583–601. Scholar
  25. 25.
    Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53(Pt 3):240–55. Scholar
  26. 26.
    Cowtan KD, Main P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr D Biol Crystallogr. 1996;52(Pt 1):43–8. Scholar
  27. 27.
    Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat Protoc. 2008;3(7):1171–9. Scholar
  28. 28.
    Sheldrick GM. A short history of SHELX. Acta Crystallogr A: Found Crystallogr. 2008;64(Pt 1):112–22. Scholar
  29. 29.
    Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486–501. Scholar
  30. 30.
    Bielnicki J, Devedjiev Y, Derewenda U, Dauter Z, Joachimiak A, Derewenda ZS. B. subtilis ykuD protein at 2.0 a resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes. Proteins. 2006;62(1):144–51. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Tan TC, Mijts BN, Swaminathan K, Patel BK, Divne C. Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch. J Mol Biol. 2008;378(4):852–70. Scholar
  32. 32.
    LaPorte SL, Juo ZS, Vaclavikova J, Colf LA, Qi X, Heller NM, et al. Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system. Cell. 2008;132(2):259–72. Scholar
  33. 33.
    Matthews DA, Alden RA, Birktoft JJ, Freer T, Kraut J. Re-examination of the charge relay system in subtilisin comparison with other serine proteases. J Biol Chem. 1977;252(24):8875–83.PubMedGoogle Scholar
  34. 34.
    Robertus JD, Kraut J, Alden RA, Birktoft JJ. Subtilisin; a stereochemical mechanism involving transition-state stabilization. Biochemistry. 1972;11(23):4293–303. Scholar
  35. 35.
    Betts JC. Transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis. IUBMB Life. 2002;53(4–5):239–42. Scholar
  36. 36.
    Keren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio. 2011;2(3):e00100–11. Scholar
  37. 37.
    Mainardi JL, Hugonnet JE, Rusconi F, Fourgeaud M, Dubost L, Moumi AN, et al. Unexpected inhibition of peptidoglycan LD-transpeptidase from enterococcus faecium by the beta-lactam imipenem. J Biol Chem. 2007;282(42):30414–22. CrossRefPubMedGoogle Scholar
  38. 38.
    Biarrotte-Sorin S, Hugonnet JE, Delfosse V, Mainardi JL, Gutmann L, Arthur M, et al. Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase. J Mol Biol. 2006;359(3):533–8. Scholar
  39. 39.
    Triboulet S, Arthur M, Mainardi JL, Veckerle C, Dubee V, Nguekam-Moumi A, et al. Inactivation kinetics of a new target of beta-lactam antibiotics. J Biol Chem. 2011;286(26):22777–84. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Triboulet S, Dubee V, Lecoq L, Bougault C, Mainardi JL, Rice LB, et al. Kinetic features of L,D-transpeptidase inactivation critical for beta-lactam antibacterial activity. PLoS One. 2013;8(7):e67831. Scholar
  41. 41.
    Fedarovich A, Nicholas RA, Davies C. Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis. J Mol Biol. 2010;398(1):54–65. Scholar
  42. 42.
    Das R, Pandey GK. Expressional analysis and role of calcium regulated kinases in abiotic stress signaling. Curr Genomics. 2010;11(1):2–13. Scholar
  43. 43.
    Wagner D, Maser J, Lai B, Cai Z, Barry CE 3rd, Honer Zu Bentrup K, et al. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J Immunol. 2005;174(3):1491–500. Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  1. 1.Division of Microbiology, National Center for Toxicological ResearchUS-FDAJeffersonUSA
  2. 2.Department of Physiology & BiophysicsUniversity of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations