Inhibition of the Clostridioides difficile Class D β-Lactamase CDD-1 by Avibactam

Abstract

Avibactam is a potent diazobicyclooctane inhibitor of class A and C β-lactamases. The inhibitor also exhibits variable activity against some class D enzymes from Gram-negative bacteria; however, its interaction with recently discovered class D β-lactamases from Gram-positive bacteria has not been studied. Here, we describe microbiological, kinetic, and mass spectrometry studies of the interaction of avibactam with CDD-1, a class D β-lactamase from the clinically important pathogen Clostridioides difficile, and show that avibactam is a potent irreversible mechanism-based inhibitor of the enzyme. X-ray crystallographic studies at three time-points demonstrate the rapid formation of a stable CDD-1-avibactam acyl–enzyme complex and highlight differences in the anchoring of the inhibitor by class D enzymes from Gram-positive and Gram-negative bacteria.

In Crystallo Time-Resolved Interaction of the Clostridioides difficile CDD-1 enzyme with Avibactam Provides New Insights into the Catalytic Mechanism of Class D β-lactamases

“…The six water molecules which remain are hydrogen bonded to the avibactam molecule. 9 Crystals of CDD-1* soaked in 0.1 M imidazole pH 7.0, 85% MPD augmented with avibactam were isomorphous with the apo-CDD-1* crystal in the same buffer. Consequently, the structures of the avibactam-CDD-1* complexes at 11 soak times (Table 1) were solved by molecular substitution using the refined apo-CDD-1* structure as the starting model.…”

“…The interaction of both the A and B avibactam conformers with the CDD-1 active site has been described in detail previously. 9 In addition to the two hydrogen bonds anchoring the O7 carbonyl oxygen in both conformers, the sulfate moiety of avibactam_A makes four hydrogen bonds with residues from strand β8, and the carboxamide group on the opposite side of the piperidine ring is hydrogen bonded to two water molecules. These interactions are similar to those observed in other class D β-lactamase complexes of avibactam.…”

“…These interactions are similar to those observed in other class D β-lactamase complexes of avibactam. 9 Upon rotation to the avibactam_B conformation, all hydrogen bonding contacts between the sulfate group and the protein are lost and replaced by interactions with water molecules only. This B conformer is unlikely to be as energetically favored as the A conformer.…”

“…30 More recently, the first report of avibactam inhibition of a Gram-positive class D enzyme, CDD-1 from C. difficile, an anaerobic Gram-positive gut pathogen that has become one of the most common causes of life-threatening nosocomial infections, was published. 9 It was observed that the interaction of the inhibitor with CDD-1 was essentially irreversible and that over time the covalently bound inhibitor adopted two conformations in the substrate binding site. The first conformation, which appeared almost instantly after soaking apo-CDD-1 crystals in avibactam, was described as the canonical conformation.…”

“…Further analysis of the structures of avibactam-CDD-1 at different soak times also suggested that the carboxylated catalytic lysine, present in the apoenzyme and at short soak times, was fully decarboxylated after a longer soak time. 9 To gain further insight into the structural changes that take place within the CDD-1 active site upon the inhibitor binding, we have undertaken a detailed in crystallo time-resolved analysis of CDD-1 inhibition by avibactam. The use of timeresolved crystallography provides an opportunity to observe the catalytic mechanism of an enzyme over time.…”