The basis for resistance to beta-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus.
Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.
Fuda
Cosimo
C
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Suvorov
Maxim
M
Vakulenko
Sergei B
SB
Mobashery
Shahriar
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002384:Catalysis; D002473:Cell Wall; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D014644:Genetic Variation; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008712:Methicillin / Q000494:pharmacology*; D008956:Models, Chemical; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry*; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000378:metabolism*; D013997:Time Factors; D047090:beta-Lactams / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D008712:Methicillin