Crystallographic snapshots of Class A {beta}-lactamase catalysis reveal structural changes that facilitate {beta}-lactam hydrolysis [Enzymology]

β-lactamases confer resistance to β-lactam-based antibiotics. There is great interest in understanding their mechanisms to enable the development of β-lactamase-specific inhibitors. The mechanism of Class A β-lactamases has been studied extensively, revealing Lys73 and Glu166 as general bases that assist the catalytic residue Ser70. However, the specific roles of these two residues within the catalytic cycle remains not fully understood. To help resolve this question, we first identified a Glu166His mutant that is functional but kinetically slowed. We then carried out time-resolved crystallographic study of a full cycle of the catalytic reaction. We obtained structures that represent apo, ES*-acylation and ES*-deacylation states and analyzed the conformational changes of His166. The "in" conformation in the apo structure allows His166 to form a hydrogen bond with Lys73. The unexpected "flipped-out" conformation of His166 in the ES*-acylation structure was further examined by molecular dynamics simulations, which suggested deprotonated Lys73 serving as the general base for acylation. The "revert-in" conformation in the ES*-deacylation structure aligns His166 toward the water molecule that hydrolyzes the acyl adduct. Finally, when the acyl adduct is fully hydrolyzed, His166 rotates back to the "in" conformation of the apo state, restoring the Lys73-His166 interaction. Using His166 as surrogate, our study identifies distinct conformational changes within the active site during catalysis. We suggest that the native Glu166 executes similar changes in a less constricted way. Taken together, this structural series improves our understanding of β-lactam hydrolysis in this important class of enzymes.

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