Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239

Abstract

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced approximately 3-fold and 20-fold respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical to that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of β-lactam drugs.

Origin of OXA-23 Variant OXA-239 from a Recently Emerged Lineage of Acinetobacter baumannii International Clone V

“…OXA-239 and OXA-469 differ only at the amino acid position 250 (M in OXA-239 and I in OXA-469), and both OXA-239 and OXA-469 had three amino acid substitutions (S109L, D222N, and P225S) compared to OXA-23. These changes have been proposed to be responsible for stabilizing the enzyme and for increasing the enzyme's substrate profile to include cephalosporins and monobactams, at the expense of a decreased ability to hydrolyze carbapenems (28). To establish which transposon carries the bla OXA-239 gene, we located the contig from each strain that carried the bla OXA-23-like gene and analyzed the upstream and downstream regions (Fig.…”

Structural Analysis of The OXA-48 Carbapenemase Bound to A “Poor” Carbapenem Substrate, Doripenem

“…Additional structural comparisons were carried out with other CHDL-doripenem complexes, OXA-239 (a variant of OXA-23) and OXA-24/40 [15,36]. Both these OXAs possess a “hydrophobic bridge” that narrows the active site significantly (comprised of OXA-23 residues: F110 and M221; OXA-24 residues: Y112 and M223); this bridge is not present in OXA-48.…”

“…The wild-type OXA-23 CHDL hydrolyzes doripenem well with a k cat / K m value of 1,600 mM −1 s −1 ; whereas OXA-239’s catalytic efficiency is ~3-fold lower at 480 mM −1 s −1 [37]. Regarding the OXA-239 complex, the tautomeric state of doripenem could not be elucidated as both Δ 1 and Δ 2 isomers equally fit the electron density [36]. The same group also determined the structure of OXA-239 complexed with imipenem and observed the Δ 1 tautomer for this carbapenem [36].…”

Intrinsic Class D β-Lactamases of Clostridium difficile

“…Although CDD-1 has a substrate profile similar to that of the only other characterized class D β-lactamase from Gram-positive bacteria, BPU-1, it has superior (up to 200-fold-higher) catalytic efficiency against expanded-spectrum cephalosporins and aztreonam (28). While the substrate profile of CDD-1 is much broader than that of mutants of OXA-2 and OXA-10 β-lactamases from Pseudomonas aeruginosa (39, 40), it is more similar to those of some mutant class D β-lactamases from Acinetobacter baumannii (41 – 44). However, as with BPU-1, the catalytic activity of CDD-1 is much higher.…”