?-Lactamases continue to pose a serious threat to patients with bacterial infections. Among the most understudied are the AmpC cephalosporinases. These class C ?-lactamases confer high-level resistance to penicillins, cephalosporins, and monobactams. Additionally, increased AmpC expression in conjunction with loss of porins (e.g. increased expression of the Pseudomonas Derived Cephalosporinase (PDC) and loss of OprD in P. aeruginosa) results in resistance to imipenem, our ?last line? agent in the treatment of these serious infections. The recent introduction of the novel ?-lactam-?-lactamase inhibitor combination, ceftolozane-tazobactam (TOL/TAZO) offered hope as TOL, a 3'-aminopyrazolium cephalosporin, eludes hydrolysis by the AmpCs and does not require the presence of OprD for cell entry. Therefore, TOL/TAZO would be an ?answer? to cephalosporin and imipenem-resistant P. aeruginosa infections. Unfortunately, descriptions of resistance to TOL/TAZO rapidly emerged. As early as 2014, AmpC variants found in P. aeruginosa (e.g. the E247K substitution in PDC) demonstrated an extended-spectrum AmpC (ESAC) phenotype and were TOL/TAZO resistant. These findings alerted us to the increasing number of ESACs that are emerging and directed our focus to overcome this challenge. Our most recent research efforts led us to discover that certain amino acid substitutions in PDC were responsible for enhanced catalytic efficiency towards TOL and other expanded- spectrum cephalosporins. Studies in our laboratory with ceftazidime/avibactam raise the concern that even the newer cephalosporins (e.g., cefiderocol) will likely meet a similar fate unless a better understanding of structure function relationships in AmpCs is achieved. In this proposal, we will investigate why the ?-loop and R2 region of PDC and other AmpCs are ?hot-spots? for these substitutions. Furthermore, we propose to study PDC variants with enhanced hydrolysis of TOL (and other cephalosporins) as a model system representative of other ESACs. Lastly, we will endeavor to explore an entirely novel approach to AmpC inhibition that relies upon conformational changes. In Aim 1 we will determine the mechanistic basis and structural evolution of PDC variants located in the ?-loop (e.g., at residues V239, G242, E247, and Y249) that confer an ESAC phenotype and resistance to TOL/TAZO. We believe this phenotype arises due to increased conformational flexibility of the ?-loop that promotes TOL hydrolysis. In Aim 2 we will probe structure-function relationships of the PDC variants using cephalosporin and TOL based boronic acid transition state inhibitors (BATSIs). We believe the PDC variants have altered acylation and deacylation transition states and these strategically designed compounds will reveal the mechanistic details of catalysis. In Aim 3 we will identify allosteric sites that are critical for the structure and function of PDC and variants. We posit that PDC variants possess allosteric sites that modulate hydrolytic activity. These fundamental insights can lead to a deeper understanding of structure activity relationships and advance the design and testing of novel compounds to overcome this resistance.