Molecular dynamics simulations of class C β-lactamase from Citrobacter freundii:: Insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys(67) residue or an anionic Tyr(150) side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr(150) state being favored by similar to 1 kcal/mol. In contrast, for the noncovalent complex formed between the C freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys(67) residue is much more stable than the anionic Tyr(150) one (similar to 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys(67) state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser(64) and the neutral amino side chain of Lys(67). Thus, both the computed free energies and the structural analyses support the assignation of Lys(67) as the base catalyst for the acylation step in the native form of the C freundii enzyme.