A transition state-like acylenzyme conformation distinguishes carbapenemase activity in class A β-lactamases

Carbapenems serve as potent beta-lactam antibiotics for treating Gram-negative infections, resisting decay from most beta-lactamases. However, a growing number of enzymes now exhibit carbapenem-hydrolyzing activity, referred to as carbapenemases. Class A beta-lactamases, characterized by active-site serine enzymes that hydrolyze beta-lactams via a covalent acylenzyme intermediate, are the most widely spread among the four recognized subclasses.

While many class A enzymes form long-lasting acylenzyme complexes with carbapenems, a subset can directly hydrolyze carbapenems.

To understand the factors behind these differences, researchers performed molecular dynamics (MD) simulations on acylenzyme complexes and tetrahedral intermediates (TI) from a collection of class A beta-lactamases. The simulations uncovered several features linked to catalytic activity among the tested enzymes, such as more extensive interactions of the carbapenem acylenzyme carbonyl and generally longer active site water molecule lifetimes for deacylation.

By examining the dynamic trajectories, the findings indicated carbapenemases have diminished root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI states, not confined to the active site. This suggests that the acylenzyme complex is pre-organized for reaction in carbapenemases, unlike carbapenem-inhibited enzymes.

Additionally, principal component analysis (PCA) of acylenzyme and TI dynamics demonstrated enhanced overlap between the two states in carbapenemases, further supporting the notion of acylenzyme pre-organization. These simulations could serve as a valuable computational tool to identify enzymes with carbapenemase activity at a relatively low computational expense.