Fig 1.
Structures of β-lactam antibiotics with positions numbered.
Fig 2.
Positions of the variants residues included in this study on the KPC-2 enzyme.
Positions that are substituted in variant enzymes are highlighted in red. The catalytic Ser70 is represented in yellow as a ball and stick model.
Table 1.
Nucleotide polymorphisms and amino acid changes in variants as compared to KPC-2.
Table 2.
Minimum inhibitory concentrations (MIC’s) of antibiotics for KPC variants.
Table 3.
Kinetic parameters of KPC variants.
Fig 3.
Progress curves of KPC-2 (black), single mutants (blue) and double mutants (red) and no enzyme control (green) for ceftazidime hydrolysis.
All reactions were performed with 500 nM enzyme and 50 μM ceftazidime. Hydrolysis of ceftazidime results in a loss of absorbance at 260 nm.
Fig 4.
Bar graph comparing the MIC for ceftazidime (black) and catalytic efficiency for ceftazidime hydrolysis (gray).
Both values are represented as fold changes compared to KPC-2.
Table 4.
Free energy values and additivity relationships between substituents for ceftazidime hydrolysis.
Fig 5.
Thermal unfolding curves of KPC variants as measured by circular dichroism at 222 nm.
The identity of each variant is indicated by the symbol shape and color shown in the inset.
Table 5.
Melting temperatures of KPC-2 and variants.
Fig 6.
Protein expression levels of KPC-2 β-lactamase and variant enzymes.
KPC-2 is represented in black, single mutants in blue and double mutants in red. Band intensities from two independent experiments were used to plot the bar graph.
Fig 7.
Molecular model of ceftazidime binding to the variant P104R:H274Y (KPC-10).
The residues are represented in cyan and ceftazidime is represented in black. The dotted lines represent hydrogen bonds with the distances labeled.
Fig 8.
Correlation plot of log catalytic efficiency for ceftazidime (y-axis) as compared to thermal stability of the variants (x-axis).
KPC-2 (black circle), Single mutants (blue circle), Double mutants (red circle).