Twelve Positions In A β-lactamase That Can Expand Its Substrate ...

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Abstract

The continuous evolution of β-lactamases resulting in bacterial resistance to β-lactam antibiotics is a major concern in public health, and yet the underlying molecular basis or the pattern of such evolution is largely unknown. We investigated the mechanics of the substrate fspectrum expansion of the class A β-lactamase using PenA of Burkholderia thailandensis as a model. By analyzing 516 mutated enzymes that acquired the ceftazidime-hydrolyzing activity, we found twelve positions with single amino acid substitutions (altogether twenty-nine different substitutions), co-localized at the active-site pocket area. The ceftazidime MIC (minimum inhibitory concentration) levels and the relative frequency in the occurrence of substitutions did not correlate well with each other, and the latter appeared be largely influenced by the intrinsic mutational biases present in bacteria. Simulation studies suggested that all substitutions caused a congruent effect, expanding the space in a conserved structure called the omega loop, which in turn increased flexibility at the active site. A second phase of selection, in which the mutants were placed under increased antibiotic pressure, did not result in a second mutation in the coding region, but a mutation that increased gene expression arose in the promoter. This result suggests that the twelve amino acid positions and their specific substitutions in PenA may represent a comprehensive repertoire of the enzyme's adaptability to a new substrate. These mapped substitutions represent a comprehensive set of general mechanical paths to substrate spectrum expansion in class A β-lactamases that all share a functional evolutionary mechanism using common conserved residues.

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Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Single amino acid substitutions in…

Figure 1. Single amino acid substitutions in PenA.

a. Map of the twelve positions and…

Figure 1. Single amino acid substitutions in PenA. a. Map of the twelve positions and twenty nine amino acid substitutions. The PenA protein is represented by an arrow, on which four conserved domains of sequences and the omega loop are indicated. The twelve positions are denoted by position numbers (numbering according to Ambler et al. [17]) and the amino acid residues are denoted using the three-letter code. Amino acid substitutions are shown above the original amino acids. The MIC levels for ceftazidime are shown in the bar graph. b. Substitution patterns in 516 mutated PenA enzymes. For each position, the amino acid residue (AA) of the original and those coded for based on a possible single-nucleotide mutation are listed with molecular weight (MW) and the number of occurrence (NO). Substitutions previously found in other class A β-lactamases, that are based on the data from the Lahey Clinic (http://www.lahey.org/Studies/), are denoted by another set of colored symbols.
Figure 2

Figure 2. Sequence conservation of the 12…

Figure 2. Sequence conservation of the 12 substituted positions in PenA among class A β-lactamases.

Figure 2. Sequence conservation of the 12 substituted positions in PenA among class A β-lactamases. PenA proteins from B. thailandensis (Bt), B. pseudomallei (Bp), and B. mallei (Bm) and representatives of the three largest families of class A β-lactamases, TEM-1, SHV-1, and CTX-M-9, are aligned. The twelve positions with changes in Bt PenA* are denoted in shades of gray. Positions 69 and 167 have residues in other β-lactamases that differ from those of B. thailandensis PenA, which are denoted with blue and red arrowheads, respectively. Three of the four conserved domains in class A β-lactamases, 70SXXK73, 130SDN132, and 166EXXLN170, are denoted by boxes on the Bt PenA sequence.
Figure 3

Figure 3. Simulated structure of wild-type PenA.

Figure 3. Simulated structure of wild-type PenA.

The twelve amino acid positions that are targets…

Figure 3. Simulated structure of wild-type PenA. The twelve amino acid positions that are targets for substitution are highlighted. a. Clustering of the twelve amino acid residues in PenA. The front, side, and back views of the simulated structure are shown. Each amino acid residue is color-coded for distinction. The predicted binding site, identified using CASTp by comparing PenA with the binding site of CTX-M-9 interacting with cefoxitin, is denoted with a red closed circular line. b. A closer look at the twelve-residue cluster. The front, side, and back views of the twelve-residues, each numbered from 1 to 12, are shown. Red boxes denote residues Cys69 (1) and Asn136 (2), which are distinct from the rest of the residues because they are not part of the omega loop. C. The omega loop structure with ten substituted residues denoted by red solid circles and the other residues by unfilled circles.
Figure 4

Figure 4. Altered distances in the omega…

Figure 4. Altered distances in the omega loop in PenA*.

The measured distances are denoted…

Figure 4. Altered distances in the omega loop in PenA*. The measured distances are denoted A, B, and C in the simulated 3D omega loop structure. a. Alteration in the distance between positions 164 and 179. The distance between positions 164 and 179, where the ionic bond is present (at least in the wild type), of each of the PenA*s is compared to that of the wild-type in a bar graph. b. Altered internal space in the omega loop in PenA*s. The sum of the distances between positions 164–173 and 163–174 in the omega loop is compared to that of the wild-type in the bar graph.
Figure 5

Figure 5. Mutations from the second-round selection…

Figure 5. Mutations from the second-round selection with an increased ceftazidime level.

a. The G→A…

Figure 5. Mutations from the second-round selection with an increased ceftazidime level. a. The G→A point mutation found in the putative promoter region of penA. Nucleotide sequences of the wild-type (E264) and the mutant (E166K1-F1) with a point mutation in the putative promoter of penA (E166K1-F1) of B. thailandensis (Bt) are shown and are aligned with multiple sequences from B. pseudomallei (Bp) and B. mallei (Bm) strains. The point mutation is denoted with a label and highlighted. The putative -10 and -35 regions are denoted in shades of red and blue, respectively, and putative start codons (ATG) are denoted with red boxes. b. Ceftazidime-hydrolyzing activities of the promoter mutant. The ceftazidime (CAZ) MIC values of a promoter mutant (E166K1-F1) and its parental strain (E166K1) are compared in the bar graph. Vector-carried penA* fragments from E166K1-F1 and E166K1 are also compared in the background of E264(ΔpenA). c. Comparison of the transcription levels of the promoter mutant (E166K1-F1) and its parental strain (E166K1). The relative expression levels measured by microarrays are shown in the graph.
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