Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane prote ins that catalyze the final steps of the peptidoglycan synthesis. Resistanc e to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBP s. S. pneumoniae PEP 2a belongs to the class A high-molecular-mass PBPs hav ing both glycosyltransferase (GT) and transpeptide (TP) activities. Structu ral and functional studies of both domains are required to unravel the mech anisms of resistance, a prerequisite for the development of novel antibioti cs. The extracellular region of S. pneumoniae PBP 2a has been expressed (PB P 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. T he acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-how fluorometry. The acylation efficiency toward benzylpenicill in was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but no t in resistance to benzylpenicillin. The TP domain was purified following l imited proteolysis. PBP 2a* required detergents for solubility and interact ed with lipid vesicles, while the TP domain was water soluble. We propose t hat PBP 2a* interacts with the cytoplasmic membrane in a region distinct fr om its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.