Antimicrobial resistance (AMR) has escalated into a global crisis, and Class C β-lactamases are serine-dependent hydrolases that contribute significantly to bacterial resistance against β-lactam antibiotics, particularly higher-generation cephalosporins. Among them, Acinetobacter-derived cephalosporinases (ADC enzymes) represent clinically important and structurally dynamic variants. These enzymes possess an expanded and flexible active site, shaped by key structural elements including the Ω-loop and a spacious R2-binding pocket, which together enable efficient accommodation and hydrolysis of bulky cephalosporin substrates. Catalysis proceeds via a conserved serine-mediated acylation–deacylation mechanism, but distinct loop dynamics and active-site organization differentiate Class C enzymes from other β-lactamase classes.
A critical determinant of substrate specificity and catalytic efficiency in ADC enzymes lies in three flexible regions: the C-loop, P2-loop, and R2-loop. Mutations within these loops drive functional diversification by modulating substrate access, positioning, and turnover. In particular, R2-loop mutations are associated with expansion of the R2-binding pocket, facilitating hydrolysis of bulky cephalosporins and, in some cases, enabling carbapenem binding. C-loop alterations influence substrate stabilization and transition-state formation, while P-loop mutations affect catalytic residue alignment and proton transfer, collectively tuning enzymatic kinetics.
Beyond β-lactam hydrolysis, certain Class-C variants exhibit promiscuous esterase activity, reflecting the inherent catalytic flexibility of the serine hydrolase fold. This secondary activity likely arises from loop-mediated adaptability that allows alternative substrate orientations within the active site. Furthermore, although Class C β-lactamases are traditionally considered weak carbapenemases, specific ADC variants acquire carbapenem hydrolysing ability through cumulative loop mutations that expand the active site and optimise catalytic water positioning.
Overall, ADC β-lactamases exemplify a structurally adaptable and evolutionarily plastic enzyme system in which mutations in the C,P2 & R2 loops drive expansion from cephalosporin specificity to broader substrate promiscuity, including esterase activity and, in certain cases, carbapenem hydrolysis. Understanding these loop-governed mechanisms provides key insights into enzyme evolution, catalytic flexibility, and the ongoing emergence of multidrug resistance.