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Journal of Bacteriology, November 1998, p. 5652-5659, Vol. 180, No. 21
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
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 1a
-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
-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.
Publication 536 of the Institut de Biologie Structurale Jean-Pierre
Ebel.
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