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. 2000 Aug 15;350(Pt 1):75–80.

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae.

S Mahapatra 1, S Bhakta 1, J Ahamed 1, J Basu 1
PMCID: PMC1221226  PMID: 10926828

Abstract

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627-4630]. PBP1-Xaa-beta-lactamase fusions generated periplasmic beta-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [DeltaM1-R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [DeltaM1-R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

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Selected References

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  1. Basu J., Mahapatra S., Kundu M., Mukhopadhyay S., Nguyen-Distèche M., Dubois P., Joris B., Van Beeumen J., Cole S. T., Chakrabarti P. 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. 1996 Mar;178(6):1707–1711. doi: 10.1128/jb.178.6.1707-1711.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowler L. D., Spratt B. G. Membrane topology of penicillin-binding protein 3 of Escherichia coli. Mol Microbiol. 1989 Sep;3(9):1277–1286. doi: 10.1111/j.1365-2958.1989.tb00278.x. [DOI] [PubMed] [Google Scholar]
  3. Broome-Smith J. K., Spratt B. G. A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology. Gene. 1986;49(3):341–349. doi: 10.1016/0378-1119(86)90370-7. [DOI] [PubMed] [Google Scholar]
  4. Broome-Smith J. K., Tadayyon M., Zhang Y. Beta-lactamase as a probe of membrane protein assembly and protein export. Mol Microbiol. 1990 Oct;4(10):1637–1644. doi: 10.1111/j.1365-2958.1990.tb00540.x. [DOI] [PubMed] [Google Scholar]
  5. Ghuysen J. M. Molecular structures of penicillin-binding proteins and beta-lactamases. Trends Microbiol. 1994 Oct;2(10):372–380. doi: 10.1016/0966-842x(94)90614-9. [DOI] [PubMed] [Google Scholar]
  6. Goffin C., Ghuysen J. M. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol Mol Biol Rev. 1998 Dec;62(4):1079–1093. doi: 10.1128/mmbr.62.4.1079-1093.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Granier B., Jamin M., Adam M., Galleni M., Lakaye B., Zorzi W., Grandchamps J., Wilkin J. M., Fraipont C., Joris B. Serine-type D-Ala-D-Ala peptidases and penicillin-binding proteins. Methods Enzymol. 1994;244:249–266. doi: 10.1016/0076-6879(94)44021-2. [DOI] [PubMed] [Google Scholar]
  8. Ishino F., Mitsui K., Tamaki S., Matsuhashi M. Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A. Biochem Biophys Res Commun. 1980 Nov 17;97(1):287–293. doi: 10.1016/s0006-291x(80)80166-5. [DOI] [PubMed] [Google Scholar]
  9. Lefèvre F., Rémy M. H., Masson J. M. Topographical and functional investigation of Escherichia coli penicillin-binding protein 1b by alanine stretch scanning mutagenesis. J Bacteriol. 1997 Aug;179(15):4761–4767. doi: 10.1128/jb.179.15.4761-4767.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lepage S., Dubois P., Ghosh T. K., Joris B., Mahapatra S., Kundu M., Basu J., Chakrabarti P., Cole S. T., Nguyen-Distèche M. Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae. J Bacteriol. 1997 Jul;179(14):4627–4630. doi: 10.1128/jb.179.14.4627-4630.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Massova I., Mobashery S. Kinship and diversification of bacterial penicillin-binding proteins and beta-lactamases. Antimicrob Agents Chemother. 1998 Jan;42(1):1–17. doi: 10.1128/aac.42.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mollerach M. E., Partoune P., Coyette J., Ghuysen J. M. Importance of the E-46-D-160 polypeptide segment of the non-penicillin-binding module for the folding of the low-affinity, multimodular class B penicillin-binding protein 5 of Enterococus hirae. J Bacteriol. 1996 Mar;178(6):1774–1775. doi: 10.1128/jb.178.6.1774-1775.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nakagawa J., Tamaki S., Tomioka S., Matsuhashi M. Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase. J Biol Chem. 1984 Nov 25;259(22):13937–13946. [PubMed] [Google Scholar]
  14. Nicholas R. A., Lamson D. R., Schultz D. E. Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor. J Biol Chem. 1993 Mar 15;268(8):5632–5641. [PubMed] [Google Scholar]
  15. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rudolph R., Lilie H. In vitro folding of inclusion body proteins. FASEB J. 1996 Jan;10(1):49–56. [PubMed] [Google Scholar]
  17. Suzuki H., van Heijenoort Y., Tamura T., Mizoguchi J., Hirota Y., van Heijenoort J. In vitro peptidoglycan polymerization catalysed by penicillin binding protein 1b of Escherichia coli K-12. FEBS Lett. 1980 Feb 11;110(2):245–249. doi: 10.1016/0014-5793(80)80083-4. [DOI] [PubMed] [Google Scholar]
  18. Terrak M., Ghosh T. K., van Heijenoort J., Van Beeumen J., Lampilas M., Aszodi J., Ayala J. A., Ghuysen J. M., Nguyen-Distèche M. The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli. Mol Microbiol. 1999 Oct;34(2):350–364. doi: 10.1046/j.1365-2958.1999.01612.x. [DOI] [PubMed] [Google Scholar]
  19. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Van Heijenoort Y., Derrien M., Van Heijenoort J. Polymerization by transglycosylation in the biosynthesis of the peptidoglycan of Escherichia coli K 12 and its inhibition by antibiotics. FEBS Lett. 1978 May 1;89(1):141–144. doi: 10.1016/0014-5793(78)80540-7. [DOI] [PubMed] [Google Scholar]
  21. Wang C. C., Schultz D. E., Nicholas R. A. Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli. Biochem J. 1996 May 15;316(Pt 1):149–156. doi: 10.1042/bj3160149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Waxman D. J., Strominger J. L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu Rev Biochem. 1983;52:825–869. doi: 10.1146/annurev.bi.52.070183.004141. [DOI] [PubMed] [Google Scholar]
  23. Wu C. Y., Alborn W. E., Jr, Flokowitsch J. E., Hoskins J., Unal S., Blaszczak L. C., Preston D. A., Skatrud P. L. Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus. J Bacteriol. 1994 Jan;176(2):443–449. doi: 10.1128/jb.176.2.443-449.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. di Guilmi A. M., Mouz N., Martin L., Hoskins J., Jaskunas S. R., Dideberg O., Vernet T. Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated. J Bacteriol. 1999 May;181(9):2773–2781. doi: 10.1128/jb.181.9.2773-2781.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

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