title: Exploring the role of a conserved class A residue in the Ω-Loop of KPC-2 β-lactamase: a mechanism for ceftazidime hydrolysis.
abstract: Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-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 β-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 μm(-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 Ω-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 Ω-loop of KPC-2.
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors: Peter S
authors: PS
authors: Levitt
authors: Krisztina M
authors: KM
authors: Papp-Wallace
authors: Magdalena A
authors: MA
authors: Taracila
authors: Andrea M
authors: AM
authors: Hujer
authors: Marisa L
authors: ML
authors: Winkler
authors: Kerri M
authors: KM
authors: Smith
authors: Yan
authors: Y
authors: Xu
authors: Michael E
authors: ME
authors: Harris
authors: Robert A
authors: RA
authors: Bonomo
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D002442:Ceftazidime / Q000737:chemistry*; D002942:Circular Dichroism; D006868:Hydrolysis; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D002442:Ceftazidime; D001618:beta-Lactamases; C547585:beta-lactamase KPC-2, Klebsiella pneumoniae