Saturation mutagenesis map of generalist versus specialist adaptations of β-lactamase to novel antibiotics.

The evolution of β-lactamase proteins is shaped by the need to maintain enzymatic activity against previously prevalent β-lactam antibiotics while expanding substrate range against new classes of antibiotics. Using saturation mutagenesis and sequence-barcoding-based quantification, we comprehensively mapped the response of the fitness landscape of TEM-1 β-lactamase, which evolved against penicillin-class antibiotics, to mutational perturbations against six diverse β-lactam antibiotics. This systematic panel of antibiotic substrates, including representatives from penicillin, cephalosporin, and monobactam classes, allowed us to classify resistance mutations into two categories. Generalist mutations conferred resistance to multiple antibiotics and were consistently restricted to three positions critical for substrate recognition and catalytic function R164, G238, and E240. These substitutions produced broad spectrum resistance through mechanisms such as expansion of the active site and improved substrate accommodation. In contrast, specialist mutations conferred resistance to only a single antibiotic and exhibited much wider positional diversity. Ceftazidime selection yielded the greatest number of distinct specialist mutations, which were frequently found in flexible or peripheral regions including the omega loop. One especially unexpected finding was the identification of the E166P variant. E166 is a catalytic residue required for deacylation during hydrolysis, and substitutions at this site are generally assumed to abolish function. However, E166P conferred a significant increase in ceftazidime resistance despite eliminating activity against penicillins. Molecular dynamics simulations and mutational analysis revealed that the E166P mutant employs an alternative catalytic mechanism, involving residue N132, rather than the canonical pathway. Together, our findings reveal, at the molecular level, how specialist mutations open up a wide range of diverse and idiosyncratic solutions at the expense of generalizability. These insights may inform strategic design of antibiotic administration protocols to systematically lower pathogenic evolvability.