Insights into β-lactamases from Burkholderia species, two phylogenetically related yet distinct resistance determinants

Abstract

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k(cat) values ranging from 0.38 ± 0.04 to 460 ± 46 s(-1) and possesses high k(cat)/k(inact) values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s(-1). Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k(cat) values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.

Keywords: Antibiotic Resistance; Antibiotics; Microbiology; Molecular Modeling; Structural Biology.

FIGURE 1.

Proposed reaction mechanism for a cephalosporin (red) with a class A serine β-lactamase (black). The carbonyl of β-lactam sits in the oxyanion hole (electrophilic center) formed by the backbone nitrogens of Ser-70 and Xaa-237. The carboxylate forms a salt bridge to a positively charged amino acid, Arg-220 with PenA and PenI. Either Lys-73 directly removes a proton from Ser-70 or Glu-166 removes a proton from a water molecule (blue), which activates the nucleophilic Ser-70 to initiate acylation. Unprotonated Ser-70 attacks the β-lactam bond forming an acyl-enzyme. A proton shuttle starting with the β-lactam and Ser-130 to Lys-73 followed by Glu-166 results in an unprotonated Glu-166. Unprotonated Glu-166 removes a proton from a water molecule (i.e. DW), which attacks the acyl-enzyme complex resulting in water being added across the bond and deacylation of the inactivated β-lactam (69, 80).

FIGURE 2.

Compounds used in this study. Antibiotics from each of the four classes of β-lactams were studied, including penicillins, AMP; the cephalosporins, THIN, nitrocefin (NCF), TAX, and TAZ; the monobactam, AZT; and the carbapenem, IMI. The tautomerization of the double bond within IMI upon β-lactamase hydrolysis to form the Δ² isoform to the Δ¹ isoform is depicted in the 3rd row. β-Lactamase inhibitors utilized in this work include CLAV, SUL, and TAZO.

FIGURE 3.

Comparisons of PenA and PenI with other β-lactamases. A, amino acid sequence alignments based on BlastP search. Amino acid sequence in black background indicates identity; gray background indicates similarity, and white background indicates differences. B, phylogenetic tree comparing PenA and PenI to prevalent β-lactamases identified by Dr. George Jacoby of the Lahey Clinic.

FIGURE 4.

Overlay of the PenA (colored) and PenI (gray) active sites highlighting the following major motifs: ⁷⁰SXXK⁷³ motif (green), ¹³⁰SDN¹³² loop (orange), ²³⁴KTG²³⁶ motif (yellow), B3 β-strand (yellow), 102–110 loop (blue), and Ω loop (red), and the approximate binding pocket (gray oval) for β-lactams and β-lactamase inhibitors.

FIGURE 5.

Crystal structures of PenA and PenI. A, overlay of active site of PenA (cyan), PenI at pH 7.5 (orange), and PenI at pH 9.5 (gray) highlighting the alternate conformations of Lys-73, Ser-130, Tyr-105, and Asn-170. B, electron density of PenA active site showing the occupancy of the DW molecule is at 100%. Also represented is the water molecule in the oxyanion hole (OAW). C, electron density of PenI, pH 7.5, active site showing the occupancy of the DW molecule is at 21.7%. Also represented is the water molecule in the oxyanion hole (OAW).

FIGURE 6.

Molecular modeling of PenA and PenI with imipenem. A, Michaelis-Menten complex of PenA (cyan) with IMI (green) shows that the carbonyl of IMI is oriented toward the oxyanion hole, the backbone amides of Ser-70 and Thr-237. B, Michaelis-Menten complex of PenI (orange) with IMI (green) reveals the different conformation of Tyr-105 compared with PenA model. C, Δ² isoform of IMI (green) with PenA (cyan) reveals the carbonyl of IMI is within the oxyanion hole and in a position favorable for deacylation. D, Δ² isoform of IMI (green) with PenI (orange) demonstrates that the carbonyl of IMI is outside of oxyanion hole. Water molecules (not shown) are present near Arg-220; thus IMI is in a favorable orientation for tautomerization of its C₂–C₃ double bond for generation of the more stable Δ¹ isoform.

FIGURE 7.

Arg at position 220 is critical for imipenem resistance and hydrolysis by PenA. A, Etests for E. coli DH10B pBC SK(+) (left), E. coli DH10B pBC SK(+) bla_PenA (middle), and E. coli DH10B pBC SK(+) bla_PenA carrying the R220G substitution (right) reveals MICs of 0.19, 1.5, and 0.25 mg/liter, respectively. B, imipenem hydrolysis was monitored at 297 nm using periplasmic extracts prepared from E. coli DH10B pBC SK(+) (black line), E. coli DH10B pBC SK(+) bla_PenA (black dotted line), and E. coli DH10B pBC SK(+) bla_PenA carrying the R220G substitution (gray striped line).