Molecular Architecture and Catalytic Mechanisms of Class D Carbapenemases: Insights into Antibiotic Resistance and Carbapenem Design.

The β-lactam class of antibiotics remains indispensable for treating bacterial infections, yet their effectiveness is increasingly compromised by the emergence of carbapenem-hydrolyzing class D β-lactamases, the principal mechanism of carbapenem resistance in Acinetobacter baumannii and an important contributor to resistance in Enterobacterales. This review focuses on the structural biology and enzymatic mechanisms of these enzymes, with particular emphasis on the post-translationally carboxylated Lys73 and the solvent-restricted pocket that encloses this residue. This pocket maintains the lysine in its catalytically essential carboxylated state, enabling it to function as the general base during both acylation and deacylation. A unique gating mechanism enables the entry of a deacylating water into the catalytic lysine pocket via the opening of a hydrophobic cap induced by the 6α-hydroxyethyl group of the carbapenem following acylation of the enzyme. These insights provide a mechanistic framework for the development of next-generation therapeutics capable of overcoming carbapenem resistance. Recently designed carbapenem derivatives perturb carboxylation of the catalytic lysine or disrupt proper positioning of the catalytic water in the active site, which severely impairs deacylation and results in potent inhibition of CHDLs.