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Journal of Bacteriology, November 1998, p. 5652-5659, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification, Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1adagger

Anne Marie Di Guilmi,1 Nicolas Mouz,1 Jean-Pierre Andrieu,1 JoAnn Hoskins,2 S. Richard Jaskunas,2 Jean Gagnon,1 Otto Dideberg,1 and Thierry Vernet1,*

Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS), 38027 Grenoble Cedex 1, France,1 and Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285-04382

Received 18 May 1998/Accepted 12 August 1998

Resistance to beta -lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both beta -lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.

* Corresponding author. Mailing address: Institut de Biologie Structurale Jean-Pierre Ebel, Laboratoire d'Ingénierie des Macromolécules, 41 Avenue des Martyrs, 38027 Grenoble Cedex 1, France. Phone: 33 04 76 88 96 81. Fax: 33 04 76 88 54 94. E-mail: vernet{at}ibs.fr.

dagger Publication 536 of the Institut de Biologie Structurale Jean-Pierre Ebel.

Journal of Bacteriology, November 1998, p. 5652-5659, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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