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.

FIGURE 1.

Chemical structures of β-lactams used in this study. On ceftazidime, R₁ and R₂ side chains are indicated. Note the bulky, oxyimino R₁ side chains characteristic of the extended spectrum cephalosporins cefotaxime, ceftazidime, and cefepime, and the monobactam aztreonam.

FIGURE 2.

A, structure of KPC-2 (Protein Data Bank code 2OV1). The nucleophile Ser-70 and residue of focus in this study, Arg-164, are indicated. Active site regions are color-coded as follows: orange, SXXK motif (residues 70–73); green, SDN loop (130–132); blue, Ω-loop (164–179); red, b3 β-strand (234–242). B, sequence alignment of the Ω-loops of KPC-2 with other class A carbapenemases (blue), penicillinases (red), and cephalosporinases (green). Note the strict conservation of Arg-164 and Asp-179. C, overlay of Ω-loops from TEM-1 (red) and KPC-2 (blue), showing the salt bridge between Arg-164 and Asp-179.

FIGURE 3.

Immunoblot for KPC-2 and, as a loading control, DNA-K, probing E. coli DH10B cells harboring pBR322-catI-bla_{KPC-2 R164X} grown in LB.

FIGURE 4.

Thermal denaturation experiment. 19 μm KPC-2 or the R164S variant was incubated with or without 250 μm ceftazidime and tested by circular dichroism at λ₂₂₀. Mean residue molar ellipticity values (MRE) were converted to a fraction of folded protein at each temperature by the formula: Fraction folded = MRE_{time point}/MRE_minimum. The calculated T_m values for KPC-2 and the R164S variant were 56 and 52 °C, respectively, whereas ceftazidime (TAZ) stabilized both proteins by 1 °C.

FIGURE 5.

Determination of protein secondary structure by circular dichroism difference spectrum. 19 μm KPC-2 (A) or the R164S variant (B) was incubated either alone or with 250 μm ceftazidime (TAZ), and CD spectra were gathered and plotted as described under “Materials and Methods.”

FIGURE 6.

Deconvoluted ESI-MS spectra of ceftazidime reaction coordinate. A and B, 19 μm KPC-2 (A) or the R164S variant (B) was incubated with ceftazidime (TAZ) at a 100:1 substrate to enzyme ratio, and reactions were quenched at 5, 15, or 60 s in 0.2% formic acid, processed, and then analyzed as described under “Materials and Methods.” All of the measurements have an error of ± 5 atomic mass units. C, ceftazidime undergoing R₂ elimination to obtain the +Δ468 adduct.

FIGURE 7.

Pre-steady-state kinetic traces of ceftazidime hydrolysis. In a stopped flow spectrophotometer, 100 μm ceftazidime was incubated with 2 and 4 μm KPC-2 or 2 and 4 μm the R164S variant, and the rate of product (P) formation (converted from absorbance readings, Δϵ₂₅₆ = −7600 m⁻¹ cm⁻¹) was followed for 2 s. Representative traces are shown.

FIGURE 8.

Molecular representations of the Michaelis-Menten complexes of ceftazidime with KPC-2 or the R164S variant. A, superimposition of the KPC-2 (beige) and R164S (green) KPC-2 models, with the Ω-loops and positions of Ser-70 highlighted. The Ω-loop demonstrates slightly increased flexibility in the R164S variant, but the spatial positions of all side chains are within 1 Å in the two models. Conversely, the Oγ of Ser-70 has shifted 2.3 Å in the model for KPC-2. B, superimposition of the models, highlighting the conformations of ceftazidime in the KPC-2 (beige) and R164S (green) models. The arrow indicates the flipping of the ceftazidime aminothiazole and di-methyl carboxyl groups in the two models. C and D, representation of the predicted interactions by DS Studio 3.1 between ceftazidime and indicated active site residues in KPC-2 (C) and the R164S variant (D), indicated by blue dotted lines. In particular, note the hydrogen bond between the main chain oxygen of Asn-170 and the R₁ carboxyl group of ceftazidime in the R164S, but not KPC-2 model. Also note the more extensive stabilization of the β-lactam carbonyl in the KPC-2, but C₃ carboxylate in the R164S variant.