Abstract
We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.
Full Text
The Full Text of this article is available as a PDF (201.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Hartman B. J., Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984 May;158(2):513–516. doi: 10.1128/jb.158.2.513-516.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jamin M., Damblon C., Bauduin-Misselyn A. M., Durant F., Roberts G. C., Charlier P., Llabres G., Frère J. M. Direct n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase. Biochem J. 1994 Jul 1;301(Pt 1):199–203. doi: 10.1042/bj3010199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Labischinski H. Consequences of the interaction of beta-lactam antibiotics with penicillin binding proteins from sensitive and resistant Staphylococcus aureus strains. Med Microbiol Immunol. 1992;181(5):241–265. doi: 10.1007/BF00198846. [DOI] [PubMed] [Google Scholar]
- Matsuhashi M., Song M. D., Ishino F., Wachi M., Doi M., Inoue M., Ubukata K., Yamashita N., Konno M. Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. J Bacteriol. 1986 Sep;167(3):975–980. doi: 10.1128/jb.167.3.975-980.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nieto M., Perkins H. R., Frère J. M., Ghuysen J. M. Fluorescence and circular dichroism studies on the Streptomyces R61 DD-carboxypeptidase-transpeptidase. Penicillin binding by the enzyme. Biochem J. 1973 Nov;135(3):493–505. doi: 10.1042/bj1350493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Roychoudhury S., Dotzlaf J. E., Ghag S., Yeh W. K. Purification, properties, and kinetics of enzymatic acylation with beta-lactams of soluble penicillin-binding protein 2a. A major factor in methicillin-resistant Staphylococcus aureus. J Biol Chem. 1994 Apr 22;269(16):12067–12073. [PubMed] [Google Scholar]
- Ryffel C., Tesch W., Birch-Machin I., Reynolds P. E., Barberis-Maino L., Kayser F. H., Berger-Bächi B. Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Gene. 1990 Sep 28;94(1):137–138. doi: 10.1016/0378-1119(90)90481-6. [DOI] [PubMed] [Google Scholar]
- Sall D. J., Kaiser R. E., Jr Characterization of the interaction between human alpha-thrombin and methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carboxylate (LY806303) using electrospray mass spectrometry and tandem mass spectrometry. J Med Chem. 1993 Aug 6;36(16):2350–2355. doi: 10.1021/jm00068a012. [DOI] [PubMed] [Google Scholar]
- Song M. D., Wachi M., Doi M., Ishino F., Matsuhashi M. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 1987 Aug 31;221(1):167–171. doi: 10.1016/0014-5793(87)80373-3. [DOI] [PubMed] [Google Scholar]
- 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]