1887

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
2019-12-14
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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. [CrossRef] [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. [CrossRef]
    [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  13. Corpet F. . ( 1988; ). Multiple sequence alignment with hierarchical clustering. . Nucleic Acids Res 16:, 10881–10890. [CrossRef] [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 issue), W116–W118. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  23. Ghuysen J.-M. . ( 1991; ). Serine beta-lactamases and penicillin-binding proteins. . Annu Rev Microbiol 45:, 37–67. [CrossRef] [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. [CrossRef] [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 issue), W500–W502. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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 9), 114–122. [CrossRef] [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. [CrossRef] [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. [CrossRef]
    [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. [CrossRef] [PubMed]
    [Google Scholar]
  39. Lüthy R. , Bowie J. U. , Eisenberg D. . ( 1992; ). Assessment of protein models with three-dimensional profiles. . Nature 356:, 83–85. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  55. Rost B. , Yachdav G. , Liu J. . ( 2004; ). The PredictProtein server. . Nucleic Acids Res 32: (Web Server issue), W321–W326. [CrossRef] [PubMed]
    [Google Scholar]
  56. Sali A. , Blundell T. L. . ( 1993; ). Comparative protein modelling by satisfaction of spatial restraints. . J Mol Biol 234:, 779–815. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  65. Taboada B. , Ciria R. , Martinez-Guerrero C. E. , Merino E. . ( 2012; ). ProOpDB: prokaryotic operon database. . Nucleic Acids Res 40: (Database issue), D627–D631. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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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