Stabilizing Mutations Enhance Evolvability of BlaC β-lactamase by Widening the Mutational Landscape

Antimicrobial resistance is fueled by the rapid evolution of β-lactamases. However, a gain of new enzyme activity often comes at the expense of reduced protein stability. This evolutionary constraint is often overcome by the acquisition of stabilizing mutations that compensate for the loss of stability invoked by new function mutations. Here, we report three stabilizing mutations (I105F, H184R, and V263I) in BlaC, a serine β-lactamase from Mycobacterium tuberculosis. Using a severely destabilized variant as a template for random mutagenesis and selection, these three mutations emerged together and were able to fully restore resistance toward the antibiotic carbenicillin. In vitro characterization shows that all three mutations increase chemical and thermal stability, which leads to elevated protein levels in the periplasm of Escherichia coli. We demonstrate that the introduction of stabilizing mutations substantially enhances the evolvability of the enzyme. These findings illustrate the important role of stabilizing mutations in enzyme evolution by alleviating function-stability trade-offs and broadening the accessible evolutionary landscape.