1887

Abstract

Penicillin-binding protein 5 (PBP5), a -carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression complements the shape and restores the lost beta-lactam resistance. PBP5 has an ‘Ω-loop’-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the ‘Ω-like’ loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants, ~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was ~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of PBP5.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000052
2015-04-01
2019-12-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/161/4/895.html?itemId=/content/journal/micro/10.1099/mic.0.000052&mimeType=html&fmt=ahah

References

  1. Banerjee S., Pieper U., Kapadia G., Pannell L. K., Herzberg O.. ( 1998; ). Role of the Ω-loop in the activity, substrate specificity, and structure of class A β-lactamase. . Biochemistry 37:, 3286–3296. [CrossRef] [PubMed]
    [Google Scholar]
  2. Baquero M. R., Bouzon M., Quintela J. C., Ayala J. A., Moreno F.. ( 1996; ). dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with dd-carboxypeptidase activity. . J Bacteriol 178:, 7106–7111.[PubMed]
    [Google Scholar]
  3. Chakraborty S.. ( 2012; ). An automated flow for directed evolution based on detection of promiscuous scaffolds using spatial and electrostatic properties of catalytic residues. . PLoS ONE 7:, e40408. [CrossRef] [PubMed]
    [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Davies C., White S. W., Nicholas R. A.. ( 2001; ). Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-Å resolution. . J Biol Chem 276:, 616–623. [CrossRef] [PubMed]
    [Google Scholar]
  8. Di Guilmi A. M., Mouz N., Pétillot Y., Forest E., Dideberg O., Vernet T.. ( 2000; ). Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to β-lactam antibiotics using electrospray ionization–mass spectrometry. . Anal Biochem 284:, 240–246. [CrossRef] [PubMed]
    [Google Scholar]
  9. 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]
  10. Frére J. M., Leyh-Bouille M., Ghuysen J. M., Nieto M., Perkins H. R.. ( 1976; ). Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces. . Methods Enzymol 45:, 610–636. [CrossRef] [PubMed]
    [Google Scholar]
  11. 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]
  12. Ghosh A. S., Melquist A. L., Young K. D.. ( 2006; ). Loss of O-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli. . FEMS Microbiol Lett 263:, 252–257. [CrossRef] [PubMed]
    [Google Scholar]
  13. 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]
  14. Ghuysen J. M.. ( 1991; ). Serine β-lactamases and penicillin-binding proteins. . Annu Rev Microbiol 45:, 37–67. [CrossRef] [PubMed]
    [Google Scholar]
  15. González-Leiza S. M., de Pedro M. A., Ayala J. A.. ( 2011; ). AmpH, a bifunctional dd-endopeptidase and dd-carboxypeptidase of Escherichia coli. . J Bacteriol 193:, 6887–6894. [CrossRef] [PubMed]
    [Google Scholar]
  16. 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]
  17. Jones D. T.. ( 1999; ). Protein secondary structure prediction based on position-specific scoring matrices. . J Mol Biol 292:, 195–202. [CrossRef] [PubMed]
    [Google Scholar]
  18. Korat B., Mottl H., Keck W.. ( 1991; ). Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition. . Mol Microbiol 5:, 675–684. [CrossRef] [PubMed]
    [Google Scholar]
  19. 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]
  20. Lineweaver H., Burk D.. ( 1934; ). The determination of enzyme dissociation constants. . J Am Chem Soc 56:, 658–666. [CrossRef]
    [Google Scholar]
  21. Louis-Jeune C., Andrade-Navarro M. A., Perez-Iratxeta C.. ( 2012; ). Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. . Proteins 80:, 374–381. [CrossRef] [PubMed]
    [Google Scholar]
  22. Lüthy R., Bowie J. U., Eisenberg D.. ( 1992; ). Assessment of protein models with three-dimensional profiles. . Nature 356:, 83–85. [CrossRef] [PubMed]
    [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Nelson D. E., Ghosh A. S., Paulson A. L., Young K. D.. ( 2002; ). Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli. . J Bacteriol 184:, 3630–3639. [CrossRef] [PubMed]
    [Google Scholar]
  27. 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]
  28. Nicola G., Fedarovich A., Nicholas R. A., Davies C.. ( 2005; a). A large displacement of the SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli. . Biochem J 392:, 55–63. [CrossRef] [PubMed]
    [Google Scholar]
  29. Nicola G., Peddi S., Stefanova M., Nicholas R. A., Gutheil W. G., Davies C.. ( 2005; b). Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation. . Biochemistry 44:, 8207–8217. [CrossRef] [PubMed]
    [Google Scholar]
  30. Nilsen T., Ghosh A. S., Goldberg M. B., Young K. D.. ( 2004; ). Branching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coli. . Mol Microbiol 52:, 1045–1054. [CrossRef] [PubMed]
    [Google Scholar]
  31. Potluri L., Karczmarek A., Verheul J., Piette A., Wilkin J. M., Werth N., Banzhaf M., Vollmer W., Young K. D. et al. ( 2010; ). Septal and lateral wall localization of PBP5, the major d,d-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment. . Mol Microbiol 77:, 300–323. [CrossRef] [PubMed]
    [Google Scholar]
  32. Rost B., Yachdav G., Liu J.. ( 2004; ). The PredictProtein server. . Nucleic Acids Res 32: ( Web Server issue), W321–W326. [CrossRef] [PubMed]
    [Google Scholar]
  33. Šali A., Blundell T. L.. ( 1993; ). Comparative protein modelling by satisfaction of spatial restraints. . J Mol Biol 234:, 779–815. [CrossRef] [PubMed]
    [Google Scholar]
  34. 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]
  35. 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]
  36. Sauvage E., Powell A. J., Heilemann J., Josephine H. R., Charlier P., Davies C., Pratt R. F.. ( 2008; ). Crystal structures of complexes of bacterial dd-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle. . J Mol Biol 381:, 383–393. [CrossRef] [PubMed]
    [Google Scholar]
  37. Stefanova M. E., Davies C., Nicholas R. A., Gutheil W. G.. ( 2002; ). pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5. . Biochim Biophys Acta 1597:, 292–300. [CrossRef] [PubMed]
    [Google Scholar]
  38. Sun T., Nukaga M., Mayama K., Braswell E. H., Knox J. R.. ( 2003; ). Comparison of β-lactamases of classes A and D: 1.5-Å crystallographic structure of the class D OXA-1 oxacillinase. . Protein Sci 12:, 82–91. [CrossRef] [PubMed]
    [Google Scholar]
  39. Urbach C., Fastrez J., Soumillion P.. ( 2008; ). A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases. . J Biol Chem 283:, 32516–32526. [CrossRef] [PubMed]
    [Google Scholar]
  40. van der Linden M. P., de Haan L., Dideberg O., Keck W.. ( 1994; ). Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli. . Biochem J 303:, 357–362.[PubMed]
    [Google Scholar]
  41. 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]
  42. 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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000052
Loading
/content/journal/micro/10.1099/mic.0.000052
Loading

Data & Media loading...

Supplements

Supplementary Data



PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error