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Volume 161, Issue 5

A putative low-molecular-mass penicillin-binding protein (PBP) of exhibits prominent physiological characteristics of -carboxypeptidase and beta-lactamase Free

Abstract

-Carboxypeptidases (-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the -CPases of mycobacteria. In this study, a putative -CPase of , MSMEG_2433 is characterized. The gene for the membrane-bound form of was cloned and expressed in in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of , which was a prominent physiological characteristic of -CPases. Moreover, expression of MSMEG_2433 elevated beta-lactam resistance in PBP deletion mutants (Δ) of , strengthening its physiology as a -CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited -CPase activity against artificial and peptidoglycan-mimetic -CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours -CPase activity, analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the , and studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both -CPase and beta-lactamase activities.

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2015-05-01
2024-04-26
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References

  1. Adachi H., Ohta T., Matsuzawa H. 1991; Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin. J Biol Chem 266:3186–3191[PubMed]
     [Google Scholar]
  2. Banerjee S., Pieper U., Kapadia G., Pannell L. K., Herzberg O. 1998; Role of the omega-loop in the activity, substrate specificity, and structure of class A β-lactamase. Biochemistry 37:3286–3296 [View Article][PubMed]
     [Google Scholar]
  3. Basu J., Chattopadhyay R., Kundu M., Chakrabarti P. 1992; Purification and partial characterization of a penicillin-binding protein from Mycobacterium smegmatis . J Bacteriol 174:4829–4832[PubMed]
     [Google Scholar]
  4. Basu J., Mahapatra S., Kundu M., Mukhopadhyay S., Nguyen-Distèche M., Dubois P., Joris B., Van Beeumen J., Cole S. T. et al. 1996; Identification and overexpression in Escherichia coli of a Mycobacterium leprae gene, pon1, encoding a high-molecular-mass class A penicillin-binding protein, PBP1. J Bacteriol 178:1707–1711[PubMed]
     [Google Scholar]
  5. Basu D., Narayankumar D., Beeumen J. V., Basu J. 1997; Characterization of a beta-lactamase from Mycobacterium smegmatis SN 2. IUBMB Life 43:557–562 [View Article]
     [Google Scholar]
  6. Bhakta S., Basu J. 2002; Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis . Biochem J 361:635–639 [View Article][PubMed]
     [Google Scholar]
  7. Bourai N., Jacobs W. R. Jr, Narayanan S. 2012; Deletion and overexpression studies on DacB2, a putative low molecular mass penicillin binding protein from Mycobacterium tuberculosis H(37)Rv. Microb Pathog 52:109–116 [View Article][PubMed]
     [Google Scholar]
  8. Carapito R., Chesnel L., Vernet T., Zapun A. 2006; Pneumococcal β-lactam resistance due to a conformational change in penicillin-binding protein 2x. J Biol Chem 281:1771–1777 [View Article][PubMed]
     [Google Scholar]
  9. Chambers H. F., Sachdeva M. J., Hackbarth C. J. 1994; Kinetics of penicillin binding to penicillin-binding proteins of Staphylococcus aureus . Biochem J 301:139–144[PubMed]
     [Google Scholar]
  10. Chowdhury C., Ghosh A. S. 2011; Differences in active-site microarchitecture explain the dissimilar behaviors of PBP5 and 6 in Escherichia coli . J Mol Graph Model 29:650–656 [View Article][PubMed]
     [Google Scholar]
  11. Chowdhury C., Nayak T. R., Young K. D., Ghosh A. S. 2010; A weak DD-carboxypeptidase activity explains the inability of PBP 6 to substitute for PBP 5 in maintaining normal cell shape in Escherichia coli . FEMS Microbiol Lett 303:76–83 [View Article][PubMed]
     [Google Scholar]
  12. Chowdhury C., Kar D., Dutta M., Kumar A., Ghosh A. S. 2012; Moderate deacylation efficiency of DacD explains its ability to partially restore beta-lactam resistance in Escherichia coli PBP5 mutant. FEMS Microbiol Lett 337:73–80 [View Article][PubMed]
     [Google Scholar]
  13. Corpet F. 1988; Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 [View Article][PubMed]
     [Google Scholar]
  14. Denome S. A., Elf P. K., Henderson T. A., Nelson D. E., Young K. D. 1999; Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis. J Bacteriol 181:3981–3993[PubMed]
     [Google Scholar]
  15. 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 Acids Res 34:Web Server issueW116–W118 [View Article][PubMed]
     [Google Scholar]
  16. Dzhekieva L., Kumar I., Pratt R. F. 2012; Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids. Biochemistry 51:2804–2811 [View Article][PubMed]
     [Google Scholar]
  17. Eun H. M., Yapo A., Petit J. F. 1978; DD-Carboxypeptidase activity of membrane fragments of Mycobacterium smegmatis. Enzymatic properties and sensitivity to beta-lactam antibiotics. Eur J Biochem 86:97–103 [View Article][PubMed]
     [Google Scholar]
  18. Fisher J. F., Mobashery S. 2009; Three decades of the class A beta-lactamase acyl-enzyme. Curr Protein Pept Sci 10:401–407 [View Article][PubMed]
     [Google Scholar]
  19. Flores A. R., Parsons L. M., Pavelka M. S. Jr 2005; Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics. Microbiology 151:521–532 [View Article][PubMed]
     [Google Scholar]
  20. Fontana R., Grossato A., Rossi L., Cheng Y. R., Satta G. 1985; Transition from resistance to hypersusceptibility to beta-lactam antibiotics associated with loss of a low-affinity penicillin-binding protein in a Streptococcus faecium mutant highly resistant to penicillin. Antimicrob Agents Chemother 28:678–683 [View Article][PubMed]
     [Google Scholar]
  21. Ghosh A. S., Young K. D. 2003; Sequences near the active site in chimeric penicillin binding proteins 5 and 6 affect uniform morphology of Escherichia coli . J Bacteriol 185:2178–2186 [View Article][PubMed]
     [Google Scholar]
  22. Ghosh A. S., Chowdhury C., Nelson D. E. 2008; Physiological functions of d-alanine carboxypeptidases in Escherichia coli . Trends Microbiol 16:309–317 [View Article][PubMed]
     [Google Scholar]
  23. Ghuysen J.-M. 1991; Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol 45:37–67 [View Article][PubMed]
     [Google Scholar]
  24. Goffin C., Ghuysen J.-M. 1998; Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol Mol Biol Rev 62:1079–1093[PubMed]
     [Google Scholar]
  25. Gupta R., Lavollay M., Mainardi J.-L., Arthur M., Bishai W. R., Lamichhane G. 2010; The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat Med 16:466–469 [View Article][PubMed]
     [Google Scholar]
  26. Guzman L.-M., Belin D., Carson M. J., Beckwith J. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130[PubMed]
     [Google Scholar]
  27. Hartman B. J., Tomasz A. 1984; Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus . J Bacteriol 158:513–516[PubMed]
     [Google Scholar]
  28. Heinig M., Frishman D. 2004; stride: a web server for secondary structure assignment from known atomic coordinates of proteins. Nucleic Acids Res 32:Web Server issueW500–W502 [View Article][PubMed]
     [Google Scholar]
  29. Henry X., Amoroso A., Coyette J., Joris B. 2010; Interaction of ceftobiprole with the low-affinity PBP 5 of Enterococcus faecium . Antimicrob Agents Chemother 54:953–955 [View Article][PubMed]
     [Google Scholar]
  30. Höltje J.-V. 1998; Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli . Microbiol Mol Biol Rev 62:181–203[PubMed]
     [Google Scholar]
  31. Jacobs C., Frère J.-M., Normark S. 1997; Cytosolic intermediates for cell wall biosynthesis and degradation control inducible β-lactam resistance in gram-negative bacteria. Cell 88:823–832 [View Article][PubMed]
     [Google Scholar]
  32. Jamin M., Damblon C., Millier S., Hakenbeck R., Frère J.-M. 1993; Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with beta-lactams. Biochem J 292:735–741[PubMed]
     [Google Scholar]
  33. Jarlier V., Gutmann L., Nikaido H. 1991; Interplay of cell wall barrier and beta-lactamase activity determines high resistance to beta-lactam antibiotics in Mycobacterium chelonae . Antimicrob Agents Chemother 35:1937–1939 [View Article][PubMed]
     [Google Scholar]
  34. Krieger E., Joo K., Lee J., Lee J., Raman S., Thompson J., Tyka M., Baker D., Karplus K. 2009; Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8. Proteins 77:Suppl 9114–122 [View Article][PubMed]
     [Google Scholar]
  35. Kumar P., Arora K., Lloyd J. R., Lee I. Y., Nair V., Fischer E., Boshoff H. I., Barry C. E. III 2012; Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium tuberculosis . Mol Microbiol 86:367–381 [View Article][PubMed]
     [Google Scholar]
  36. 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 [View Article]
     [Google Scholar]
  37. Lepage S., Dubois P., Ghosh T. K., Joris B., Mahapatra S., Kundu M., Basu J., Chakrabarti P., Cole S. T. et al. 1997; Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae . J Bacteriol 179:4627–4630[PubMed]
     [Google Scholar]
  38. Leyh-Bouille M., Nguyen-Distèche M., Ghuysen J. M. 1981; On the DD-carboxypeptidase enzyme system of Streptomyces strain K15. Eur J Biochem 115:579–584 [View Article][PubMed]
     [Google Scholar]
  39. Lüthy R., Bowie J. U., Eisenberg D. 1992; Assessment of protein models with three-dimensional profiles. Nature 356:83–85 [View Article][PubMed]
     [Google Scholar]
  40. Malhotra K. T., Nicholas R. A. 1992; Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes d-alanine carboxypeptidase activity without affecting penicillin binding. J Biol Chem 267:11386–11391[PubMed]
     [Google Scholar]
  41. Massova I., Mobashery S. 1998; Kinship and diversification of bacterial penicillin-binding proteins and β-lactamases. Antimicrob Agents Chemother 42:1–17[PubMed] [CrossRef]
     [Google Scholar]
  42. McGuffin L. J., Bryson K., Jones D. T. 2000; The psipred protein structure prediction server. Bioinformatics 16:404–405 [View Article][PubMed]
     [Google Scholar]
  43. Mukherjee T., Basu D., Mahapatra S., Goffin C., van Beeumen J., Basu J. 1996; Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis . Biochem J 320:197–200[PubMed]
     [Google Scholar]
  44. Mukhopadhyay S., Chakrabarti P. 1997; Altered permeability and beta-lactam resistance in a mutant of Mycobacterium smegmatis . Antimicrob Agents Chemother 41:1721–1724[PubMed]
     [Google Scholar]
  45. Murray C. J., Ortblad K. F., Guinovart C. et al. 2014; Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 384:1005–1070 [View Article][PubMed]
     [Google Scholar]
  46. Navratna V., Nadig S., Sood V., Prasad K., Arakere G., Gopal B. 2010; Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance. J Bacteriol 192:134–144 [View Article][PubMed]
     [Google Scholar]
  47. Nelson D. E., Young K. D. 2000; Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli . J Bacteriol 182:1714–1721 [View Article][PubMed]
     [Google Scholar]
  48. Nelson D. E., Young K. D. 2001; Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli . J Bacteriol 183:3055–3064 [View Article][PubMed]
     [Google Scholar]
  49. Nemmara V. V., Dzhekieva L., Sarkar K. S., Adediran S. A., Duez C., Nicholas R. A., Pratt R. F. 2011; Substrate specificity of low-molecular mass bacterial DD-peptidases. Biochemistry 50:10091–10101 [View Article][PubMed]
     [Google Scholar]
  50. Nguyen-Distèche M., Leyh-Bouille M., Ghuysen J.-M. 1982; Isolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive d-alanyl-d-alanine-cleaving transpeptidase. Biochem J 207:109–115[PubMed]
     [Google Scholar]
  51. Nicholas R. A., Krings S., Tomberg J., Nicola G., Davies C. 2003; Crystal structure of wild-type penicillin-binding protein 5 from Escherichia coli: implications for deacylation of the acyl-enzyme complex. J Biol Chem 278:52826–52833 [View Article][PubMed]
     [Google Scholar]
  52. Normark S. 1995; β-Lactamase induction in gram-negative bacteria is intimately linked to peptidoglycan recycling. Microb Drug Resist 1:111–114 [View Article][PubMed]
     [Google Scholar]
  53. Olsson O., Bergström S., Lindberg F. P., Normark S. 1983; ampC beta-lactamase hyperproduction in Escherichia coli: natural ampicillin resistance generated by horizontal chromosomal DNA transfer from Shigella . Proc Natl Acad Sci U S A 80:7556–7560 [View Article][PubMed]
     [Google Scholar]
  54. Patru M.-M., Pavelka M. S. Jr 2010; A role for the class A penicillin-binding protein PonA2 in the survival of Mycobacterium smegmatis under conditions of nonreplication. J Bacteriol 192:3043–3054 [View Article][PubMed]
     [Google Scholar]
  55. Rost B., Yachdav G., Liu J. 2004; The PredictProtein server. Nucleic Acids Res 32:Web Server issueW321–W326 [View Article][PubMed]
     [Google Scholar]
  56. Sali A., Blundell T. L. 1993; Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815 [View Article][PubMed]
     [Google Scholar]
  57. Sandlin R. C., Goldberg M. B., Maurelli A. T. 1996; Effect of O side-chain length and composition on the virulence of Shigella flexneri 2a. Mol Microbiol 22:63–73 [View Article][PubMed]
     [Google Scholar]
  58. Sarkar S. K., Chowdhury C., Ghosh A. S. 2010; Deletion of penicillin-binding protein 5 (PBP5) sensitises Escherichia coli cells to β-lactam agents. Int J Antimicrob Agents 35:244–249 [View Article][PubMed]
     [Google Scholar]
  59. Sarkar S. K., Dutta M., Chowdhury C., Kumar A., Ghosh A. S. 2011; PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli . Microbiology 157:2702–2707 [View Article][PubMed]
     [Google Scholar]
  60. Sauvage E., Kerff F., Terrak M., Ayala J. A., Charlier P. 2008; The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32:234–258 [View Article][PubMed]
     [Google Scholar]
  61. Smith J. D., Kumarasiri M., Zhang W., Hesek D., Lee M., Toth M., Vakulenko S., Fisher J. F., Mobashery S., Chen Y. 2013; Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in β-lactam resistance. Antimicrob Agents Chemother 57:3137–3146 [View Article][PubMed]
     [Google Scholar]
  62. Sorci L., Brunetti L., Cialabrini L., Mazzola F., Kazanov M. D., D’Auria S., Ruggieri S., Raffaelli N. 2014; Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases. FEBS Lett 588:1016–1023 [View Article][PubMed]
     [Google Scholar]
  63. Stec B., Holtz K. M., Wojciechowski C. L., Kantrowitz E. R. 2005; Structure of the wild-type TEM-1 beta-lactamase at 1.55 A and the mutant enzyme Ser70Ala at 2.1 A suggest the mode of noncovalent catalysis for the mutant enzyme. Acta Crystallogr D Biol Crystallogr 61:1072–1079 [View Article][PubMed]
     [Google Scholar]
  64. Stubbs K. A., Balcewich M., Mark B. L., Vocadlo D. J. 2007; Small molecule inhibitors of a glycoside hydrolase attenuate inducible AmpC-mediated β-lactam resistance. J Biol Chem 282:21382–21391 [View Article][PubMed]
     [Google Scholar]
  65. Taboada B., Ciria R., Martinez-Guerrero C. E., Merino E. 2012; ProOpDB: prokaryotic operon database. Nucleic Acids Res 40:Database issueD627–D631 [View Article][PubMed]
     [Google Scholar]
  66. Thompson J. D., Gibson T., Higgins D. G. 2002) Multiple sequence alignment using clustal w and clustal_x . Curr Protoc Bioinformatics 2:2.3.1–2.3.22
     [Google Scholar]
  67. Vakulenko S. B., Taibi-Tronche P., Tóth M., Massova I., Lerner S. A., Mobashery S. 1999; Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) β-lactamase from Escherichia coli . J Biol Chem 274:23052–23060 [View Article][PubMed]
     [Google Scholar]
  68. Wang F., Cassidy C., Sacchettini J. C. 2006; Crystal structure and activity studies of the Mycobacterium tuberculosis beta-lactamase reveal its critical role in resistance to beta-lactam antibiotics. Antimicrob Agents Chemother 50:2762–2771 [View Article][PubMed]
     [Google Scholar]
  69. Wilkinson A.-S., Ward S., Kania M., Page M. G., Wharton C. W. 1999; Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii β-lactamase: a time-resolved FTIR spectroscopic study. Biochemistry 38:3851–3856 [View Article][PubMed]
     [Google Scholar]
  70. Wilkinson A.-S., Bryant P. K., Meroueh S. O., Page M. G., Mobashery S., Wharton C. W. 2003; A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii β-lactamase revealed by infrared spectroscopy and molecular dynamics simulations. Biochemistry 42:1950–1957 [View Article][PubMed]
     [Google Scholar]
  71. World Health Organization 2013 Global Tuberculosis Report 2013 Geneva: World Health Organization;
     [Google Scholar]
  72. Zhang W., Shi Q., Meroueh S. O., Vakulenko S. B., Mobashery S. 2007; Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli . Biochemistry 46:10113–10121 [View Article][PubMed]
     [Google Scholar]
  73. Zhao G., Meier T. I., Kahl S. D., Gee K. R., Blaszczak L. C. 1999; BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins. Antimicrob Agents Chemother 43:1124–1128[PubMed]
     [Google Scholar]
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A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase
Microbiology 161, 1081 (2015); https://doi.org/10.1099/mic.0.000074
/content/journal/micro/10.1099/mic.0.000074
/content/journal/micro/10.1099/mic.0.000074
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