Exploring the Role of a Conserved Class A Residue in the Ω-Loop of KPC-2 β-Lactamase A MECHANISM FOR CEFTAZIDIME HYDROLYSIS

Gram-negative bacteria harboring KPC-2, a class A beta-lactamase, are resistant to all beta-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A beta-lactamases and is located in the solvent-exposed Omega-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32 -> 128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative beta-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K-m for ceftazidime (0.015 -> 0.019 mu M-1 s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Omega-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through covalent trapping of the substrate by a deacylation impaired enzyme with a lower K-m. Future antibiotic design must consider the distinctive behavior of the Omega-loop of KPC-2.