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Journal of Bacteriology, March 2005, p. 2215-2217, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2215-2217.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Kinetic Characterization of the Glycosyltransferase Module of Staphylococcus aureus PBP2

Dianah Barrett,1 Catherine Leimkuhler,1 Lan Chen,2 Deborah Walker,3 Daniel Kahne,1 and Suzanne Walker2*

Department of Chemistry and Chemical Biology, Harvard University, Cambridge,1 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts,2 Department of Chemistry, Princeton University, Princeton, New Jersey3

Received 27 September 2004/ Accepted 20 December 2004


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ABSTRACT
 
We report the heterologous overexpression and purification of Staphylococcus aureus PBP2 and demonstrate efficient glycan polymerization from lipid II in vitro. S. aureus PBP2 is the first purified gram-positive class A penicillin-binding protein to show good transglycosylase activity. This enables further studies on this important class of enzymes.


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TEXT
 
Bacterial cells are surrounded by layers of peptidoglycan, a cross-linked carbohydrate polymer that protects the cells from lysing under high internal osmotic pressures (2). The glycan chains of peptidoglycan are assembled from repeating ß-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units by periplasmic glycosyltransferases (Fig. 1). Glycosyltransferases (GTs)that synthesize peptidoglycan come in two forms: as N-terminal modules in bifunctional proteins that also contain C-terminal transpeptidase modules (called class A penicillin-binding proteins [PBPs]) and as monofunctional enzymes (called monofunctional glycosyltransferases) (6, 14). These periplasmic GTs are perhaps the least understood of any of the enzymes involved in the biosynthetic pathway to peptidoglycan.



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  		FIG. 1. Chemical reaction proposed to be catalyzed by peptidoglycan glycosyltransferases. The lipid II monomeric unit is believed to add to the reducing end of the growing glycan chain (5, 14, 17). For the enzymatic reactions described in this paper, the lipid II substrate contains a heptaprenyl chain and functions as both the donor and the primer in the initial reaction cycle (3).

The paucity of information about the kinetic behavior of peptidoglycan GTs is due primarily to the fact that lipid II, the substrate needed to monitor enzymatic activity, is present in only a few thousand copies per bacterial cell and cannot be isolated readily from natural sources (15). We (18) and others (1a, 13, 16) have developed approaches to the synthesis of lipid II and analogues and have used these synthetic substrates to characterize Escherichia coli PBP1b (3, 12). Until now, however, efforts to characterize the enzymatic activity of periplasmic GTs from gram-positive organisms have not been successful (4). Here we describe the expression, purification, and characterization of Staphylococcus aureus PBP2. We chose this enzyme because resistant S. aureus strains have become a major global health care problem (7, 8), and in resistant S. aureus strains the glycosyltransferase domain of PBP2 functions together with PBP2a, an acquired methicillin-resistant transpeptidase, to make peptidoglycan (10). The glycosyltransferase module of PBP2 is thus an important antibacterial target in a major pathogen.

The pbp2 gene was PCR amplified from S. aureus (209P) purified genomic DNA by using the following primer pair: 5'-GCGCTAGCATGACGGAAAACAAAGGATCT-3' and 5'-GCGGATCCTTACTCGAGGTTGAATATACCTGTTAATCC-3'. The PCR product was subcloned into pET21b(+) (Novagen) for expression in E. coli BL21(DE3) (Novagen) as a C-terminal His6 fusion. PBP2 was expressed after induction of a log-phase culture with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 4 h at 37°C. The cells were lysed with a French pressure cell at 16,000 lb/in2 in buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) supplemented with 360 kU of rLysozyme (Novagen). Freely soluble proteins were decanted, and the pellet was resuspended in buffer containing 0.5% Sarkosyl to release membrane-anchored PBP2. The solubilized fusion protein was then loaded onto a Ni2+ column, which was washed with an imidazole gradient (5 to 60 mM) using detergent-free buffer A (20 mM Tris-HCl, 500 mM NaCl, pH 7.9) and then eluted with buffer A containing 100 to 200 mM imidazole. The protein was more than 85% pure, and the yield was estimated to be 20 mg/liter.

Enzymatic activity of the purified protein was evaluated by monitoring the incorporation of [14C]GlcNAc-labeled C35 lipid II, prepared as previously described (14), into peptidoglycan in the presence of penicillin G (1,000 U/ml), which inactivates the transpeptidase domain (3). Reactions were carried out at substrate concentrations varying from 0.2 to 16 µM in a wide-range pH buffer containing 50 mM CHES (2-[cyclohexylamino]ethanesulfonic acid), 50 mM HEPES, 50 mM acetic acid, 50 mM MES (2-morpholineethanesulfonic acid), 10 mM CaCl2, and 20% dimethyl sulfoxide. Reactions were initiated by adding enzyme to a final concentration of 90 nM as determined by titration with moenomycin, which is presumed to be a stoichiometric inhibitor of active peptidoglycan glycosyltransferases (3). The reactions were then quenched and analyzed as previously described (3, 18). Michaelis-Menten analysis (Fig. 2) established the catalytic parameters of S. aureus PBP2 under optimal conditions (pH 5.0) to be the following: Km = (4.0 ± 1.0) x 10–6 M, kcat = (1.5 ± 0.1) x 10–2 s–1, and kcat/Km = 3.4 x 103 M–1 s–1. The catalytic efficiency of S. aureus PBP2 is within 10-fold that of E. coli PBP1b (kcat/Km = 3.5 x 104 M–1 s–1) (3) and is more than 6 orders of magnitude higher than that of the only other gram-positive GT for which any kinetics have been reported, a truncated Streptococcus pneumoniae construct studied by Di Guilmi et al. (4). It should be noted that the lipid II analogue used in the present studies lacks the pentaglycine branch characteristic of S. aureus peptidoglycan precursors (11), and it is possible that the catalytic activity would be even higher in the presence of this pentaglycine substrate.



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  		FIG. 2. Velocity versus lipid II concentration for S. aureus at pH 5.0. Curve fits and associated errors were generated in Kaleidagraph using the following equation: vobs/[enzyme] = kcat x [lipid II]/(Km + [lipid II]).

There are several differences between the S. aureus enzyme and E. coli PBP1b, which is the most well-studied periplasmic GT. Notably, the two enzymes have considerably different pH optima (4.5 to 5.5 for S. aureus PBP2 versus 7.5 to 8.0 for E. coli PBP1b [12]). Second, E. coli PBP1b functions optimally in the presence of the detergent octaethylene glycol monodecyl ether (3, 9, 12), whereas the activity of the S. aureus enzyme decreased by 5- to 10-fold in the presence of all of the detergents we examined, which included octaethylene glycol monodecyl ether (0.19 mM), CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; 8.9 mM}, Sarkosyl (17.0 mM), n-octyl-ß-D-glucoside (27.0 mM), and Triton X-100 (0.77 mM). Since we used the same C35 lipid II substrate to study both PBP1b and PBP2, the different detergent requirements reflect properties of the enzymes themselves. In general, we observe that the S. aureus enzyme is more soluble and better behaved than E. coli PBP1b, which may make it a better candidate for structural and mechanistic analysis. Finally, the S. aureus enzyme is less sensitive to metal ions than the E. coli enzyme, which is strongly activated (>10-fold) by Ca2+ (12). It has been proposed that Ca2+ ions play a role in the catalytic activity of E. coli PBP1b. However, the enzymatic activity of S. aureus PBP2 is only modestly enhanced (two- to threefold) in the presence of 10 mM Ca2+, Mg2+, or Mn2+ and is not affected by the presence of 2 to 5 mM EDTA. It remains to be seen whether the significant differences in pH optima and metal ion requirements between periplasmic GTs reflect substantial differences in active site architecture that will affect the discovery of broad-spectrum GT inhibitors.

The work reported here lays the groundwork for further detailed investigations. Using the full-length S. aureus PBP2 construct as a standard, the expression and activity of truncated constructs lacking the transmembrane or transpeptidase domain can now be assessed to identify suitable candidates for structural analysis (1). Since it is unlikely that this S. aureus enzyme is unique in being well behaved, it should not be long before other active gram-positive enzymes are obtained and structures become available.


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ACKNOWLEDGMENTS
 
This work was supported by the National Institutes of Health (NIH grants R01AI50855 to S.W. and R01GM66174 to D.K.).

We thank Kim Specht for her efforts in the plasmid construction of pET21b::pbp2.


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FOOTNOTES
 
* Corresponding author. Mailing Address: Department of Microbiology & Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-5488. Fax: (617) 496-0215. E-mail: suzanne_walker{at}hms.harvard.edu. Back


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REFERENCES
 
    1
  1. Barrett, D. S., L. Chen, N. K. Litterman, and S. Walker. 2004. Expression and characterization of the isolated glycosyltransferase module of Escherichia coli PBP1b. Biochemistry 43:12375-12381.[CrossRef][Medline]
    2. 1
  2. Breukink, E., H. E. van Heusden, P. J. Vollmerhaus, E. Swiezewska, L. Brunner, S. Walker, A. J. Heck, and B. de Kruijff. 2003. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J. Biol. Chem. 278:19898-19903.[Abstract/Free Full Text]
    3. 2
  3. Bugg, T. D. 1999. Bacterial peptidoglycan biosynthesis and its inhibition, p. 241-294. In B. M. Pinto (ed.), Comprehensive natural products chemistry, vol. 3. Elsevier, Oxford, United Kingdom.
    4. 3
  4. Chen, L., D. Walker, B. Sun, Y. Hu, S. Walker, and D. Kahne. 2003. Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate. Proc. Natl. Acad. Sci. USA 100:5658-5663.[Abstract/Free Full Text]
    5. 4
  5. Di Guilmi, A. M., A. Dessen, O. Dideberg, and T. Vernet. 2003. The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains. J. Bacteriol. 185:4418-4423.[Abstract/Free Full Text]
    6. 5
  6. Fuchs-Cleveland, E., and C. Gilvarg. 1976. Oligomeric intermediate in peptidoglycan biosynthesis in Bacillus megaterium. Proc. Natl. Acad. Sci. USA 73:4200-4204.[Abstract/Free Full Text]
    7. 6
  7. Goffin, C., and J. M. Ghuysen. 1998. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62:1079-1093.[Abstract/Free Full Text]
    8. 7
  8. Lowy, F. D. 2003. Antimicrobial resistance: the example of Staphylococcus aureus. J. Clin. Investig. 111:1265-1273.[CrossRef][Medline]
    9. 8
  9. Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339:520-532.[Free Full Text]
    10. 9
  10. Nakagawa, J., S. Tamaki, S. Tomioka, and M. Matsuhashi. 1984. 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. 259:13937-13946.[Abstract/Free Full Text]
    11. 10
  11. Pinho, M. G., H. de Lencastre, and A. Tomasz. 2001. An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci. Proc. Natl. Acad. Sci. USA 98:10886-10891.[Abstract/Free Full Text]
    12. 11
  12. Schneider, T., M. M. Senn, B. Berger-Bachi, A. Tossi, H. G. Sahl, and I. Wiedemann. 2004. In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus. Mol. Microbiol. 53:675-685.[CrossRef][Medline]
    13. 12
  13. Schwartz, B., J. A. Markwalder, S. P. Seitz, Y. Wang, and R. L. Stein. 2002. A kinetic characterization of the glycosyltransferase activity of Escherichia coli PBP1b and development of a continuous fluorescence assay. Biochemistry 41:12552-12561.[CrossRef][Medline]
    14. 13
  14. Schwartz, B., J. A. Markwalder, and Y. Wang. 2001. Lipid II: total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Escherichia coli. J. Am. Chem. Soc. 123:11638-11643.[CrossRef][Medline]
    15. 14
  15. van Heijenoort, J. 2001. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 11:25R-36R.[Abstract/Free Full Text]
    16. 15
  16. van Heijenoort, Y., M. Gomez, M. Derrien, J. Ayala, and J. van Heijenoort. 1992. Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J. Bacteriol. 174:3549-3557.[Abstract/Free Full Text]
    17. 16
  17. VanNieuwenhze, M. S., S. C. Mauldin, M. Zia-Ebrahimi, B. E. Winger, W. J. Hornback, S. L. Saha, J. A. Aikins, and L. C. Blaszczak. 2002. The first total synthesis of lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis. J. Am. Chem. Soc. 124:3656-3660.[CrossRef][Medline]
    18. 17
  18. Ward, J. B., and H. R. Perkins. 1973. The direction of glycan synthesis in a bacterial peptidoglycan. Biochem. J. 135:721-728.[Medline]
    19. 18
  19. Ye, X. Y., M. C. Lo, L. Brunner, D. Walker, D. Kahne, and S. Walker. 2001. Better substrates for bacterial transglycosylases. J. Am. Chem. Soc. 123:3155-3156.[CrossRef][Medline]

Journal of Bacteriology, March 2005, p. 2215-2217, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2215-2217.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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