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. 1992 Jul;174(14):4829–4832. doi: 10.1128/jb.174.14.4829-4832.1992

Purification and partial characterization of a penicillin-binding protein from Mycobacterium smegmatis.

J Basu 1, R Chattopadhyay 1, M Kundu 1, P Chakrabarti 1
PMCID: PMC206282  PMID: 1624470

Abstract

Penicillin-binding proteins (PBPs), although characterized from several organisms, have so far not been studied in mycobacteria. The present study is the first characterization of a PBP from Mycobacterium smegmatis. The PBP was purified by solubilization of the membranes with Triton X-100 and successive chromatography of the solubilized proteins on ampicillin-linked CH Sepharose 4B and DE-52. The purified PBP (M(r), 49,500) catalyzed a model transpeptidase reaction with the tripeptide acetyl2-L-Lys-D-Ala-D-Ala as the substrate and Gly-Gly as the acceptor. The transpeptidase activity was inhibited by 50% at a benzylpenicillin concentration of 1.8 x 10(-7) M, which was similar to the concentration (1.1 x 10(-7) M) of benzylpenicillin required to saturate to 50% this PBP. Of several antibiotics tested, the concentration of antibiotic required to inhibit [35S]penicillin binding by 90% was found to be the lowest for cefoxitin and Sch 34343.

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Selected References

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  1. Coyette J., Ghuysen J. M., Fontana R. Solubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC9790. Properties of the purified enzyme. Eur J Biochem. 1978 Jul 17;88(1):297–305. doi: 10.1111/j.1432-1033.1978.tb12450.x. [DOI] [PubMed] [Google Scholar]
  2. Das P. R., Pramanik B. N., Girijavallabhan V. M., Ganguly A. K. Ionization of penem beta-lactam antibiotics using thermospray (filament-on) mass spectrometry. J Antibiot (Tokyo) 1988 Sep;41(9):1268–1271. doi: 10.7164/antibiotics.41.1268. [DOI] [PubMed] [Google Scholar]
  3. Dowson C. G., Hutchison A., Spratt B. G. Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae. Mol Microbiol. 1989 Jan;3(1):95–102. doi: 10.1111/j.1365-2958.1989.tb00108.x. [DOI] [PubMed] [Google Scholar]
  4. Dowson C. G., Hutchison A., Woodford N., Johnson A. P., George R. C., Spratt B. G. Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5858–5862. doi: 10.1073/pnas.87.15.5858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eun H. M., Yapo A., Petit J. F. DD-Carboxypeptidase activity of membrane fragments of Mycobacterium smegmatis. Enzymatic properties and sensitivity to beta-lactam antibiotics. Eur J Biochem. 1978 May;86(1):97–103. doi: 10.1111/j.1432-1033.1978.tb12288.x. [DOI] [PubMed] [Google Scholar]
  6. Frére J. M., Leyh-Bouille M., Ghuysen J. M., Nieto M., Perkins H. R. Exocellular DD-carboxypeptidases-transpeptidases from Streptomyces. Methods Enzymol. 1976;45:610–636. doi: 10.1016/s0076-6879(76)45054-1. [DOI] [PubMed] [Google Scholar]
  7. Ghuysen J. M. Bacterial active-site serine penicillin-interactive proteins and domains: mechanism, structure, and evolution. Rev Infect Dis. 1988 Jul-Aug;10(4):726–732. doi: 10.1093/clinids/10.4.726. [DOI] [PubMed] [Google Scholar]
  8. Ghuysen J. M. Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol. 1991;45:37–67. doi: 10.1146/annurev.mi.45.100191.000345. [DOI] [PubMed] [Google Scholar]
  9. Hakenbeck R., Briese T., Chalkley L., Ellerbrok H., Kalliokoski R., Latorre C., Leinonen M., Martin C. Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcus pneumoniae. J Infect Dis. 1991 Aug;164(2):313–319. doi: 10.1093/infdis/164.2.313. [DOI] [PubMed] [Google Scholar]
  10. Matsuhashi M., Wachi M., Ishino F. Machinery for cell growth and division: penicillin-binding proteins and other proteins. Res Microbiol. 1990 Jan;141(1):89–103. doi: 10.1016/0923-2508(90)90101-u. [DOI] [PubMed] [Google Scholar]
  11. Nguyen-Distèche M., Leyh-Bouille M., Ghuysen J. M. 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. 1982 Oct 1;207(1):109–115. doi: 10.1042/bj2070109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Piras G., el Kharroubi A., van Beeumen J., Coeme E., Coyette J., Ghuysen J. M. Characterization of an Enterococcus hirae penicillin-binding protein 3 with low penicillin affinity. J Bacteriol. 1990 Dec;172(12):6856–6862. doi: 10.1128/jb.172.12.6856-6862.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shepard C. C., Van Landingham R. M., Walker L. L., Good R. C. Activity of selected beta-lactam antibiotics against Mycobacterium leprae. Int J Lepr Other Mycobact Dis. 1987 Jun;55(2):322–327. [PubMed] [Google Scholar]
  14. Spratt B. G., Zhang Q. Y., Jones D. M., Hutchison A., Brannigan J. A., Dowson C. G. Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8988–8992. doi: 10.1073/pnas.86.22.8988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Utsui Y., Yokota T. Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1985 Sep;28(3):397–403. doi: 10.1128/aac.28.3.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Waxman D. J., Strominger J. L. Cleavage of a COOH-terminal hydrophobic region from D-alanine carboxypeptidase, a penicillin-sensitive bacterial membrane enzyme. Characterization of active, water-soluble fragments. J Biol Chem. 1979 Jun 10;254(11):4863–4875. [PubMed] [Google Scholar]
  17. 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]
  18. Wientjes F. B., Nanninga N. On the role of the high molecular weight penicillin-binding proteins in the cell cycle of Escherichia coli. Res Microbiol. 1991 Feb-Apr;142(2-3):333–344. doi: 10.1016/0923-2508(91)90049-g. [DOI] [PubMed] [Google Scholar]

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