The World Health organization describes antibiotic resistance as "...one of the biggest threats to global health, food security, and development today." Antibiotic resistance arises naturally and evolved long ago, but its emergence and dissemination have been considerably accelerated by the current excessive use of antibacterial drugs. This evolving resistance not only complicates standard medical practices, but also has additional expensive implications e.g. for the global economy and food production. Moreover, we are currently living in the so-called antibiotic discovery void where discovering new and safe antibacterials, especially for Gram-negative bacteria, is difficult, time-consuming, and often unprofitable for big pharmaceutical companies. β-Lactam antibiotics offer broad-spectrum antibacterial activity against Gram-negative bacteria and remain the most prescribed drugs in clinical practice. The importance of β-lactams in healthcare has been highlighted by the World Health Organization, which includes multiple different β-lactam antibiotics in their Model List of Essential Medicine. All of these antibiotics contain a four-membered β-lactam ring, which ensures antibiotic binding to penicillin-binding proteins and consequently inhibition of bacterial cell wall biosynthesis. Clinically used β-lactam compounds can be divided into four different groups: penicillins, cephalosporins, carbapenems, and monobactams, of which carbapenems play a critical role as potent antibiotics reserved for the most serious Gram-negative infections where alternatives are limited. Emerging resistance against β-lactams is evident, and especially in Gram-negative bacteria, βlactamase enzymes are the main resistance mechanism against these drugs. 17 β-Lactamases block antibiotic action by hydrolysing the β-lactam ring, which impairs efficient antibiotic binding to their ultimate target in cells. The Ambler sequence-based classification divides β-lactamases into four major subgroups: serine-β-lactamases (SBLs) comprising classes A, C, and D; and metallo-βlactamases (MBLs), class B. The hydrolysis mechanism differs between SBLs and MBLs, as SBLs utilise a nucleophilic serine residue and MBLs employ zinc cofactors. Class D SBLs are referred to as OXA (oxacillinase) enzymes, stemming from their activity against the isoxazolyl penicillin oxacillin, and they are currently of interest due to their wide distribution and the ability of many members of the group to inactivate carbapenems. The OXA enzymes include five subgroups of recognised carbapenemases: the OXA-23, OXA24/40, OXA-51, and OXA-58 βlactamases are mainly found in Acinetobacter baumannii, while OXA-48-like β-lactamases are mostly encountered in Enterobacterales. In Enterobacterales, OXA-48 β-lactamases are among the most commonly present carbapenemases in clinical samples. Their activity is relatively specific towards imipenem, but other carbapenem substrates (such as meropenem and ertapenem) are also hydrolysed, albeit slowly. The specific origin of this imipenemase activity is not well established, even though variations in measured hydrolysis rates between point variants of OXA-48 hint at structural moieties contributing to specific hydrolytic phenotypes (Figure ). In OXA-163, a partial deletion of the β5-β6 loop (Arg214-Pro217) and one amino acid substitution (Ser212Asp) expands the hydrolysis profile to accommodate expanded-spectrum oxyimino cephalosporins (such as ceftazidime) at the expense of efficient imipenem breakdown. Further studies show that the β5-β6 loop plays a role in acquired carbapenemase activity, as engineering the OXA-48 β5-β6 loop into the noncarbapenemase OXA-10 enhances its carbapenemase activity. Conversely, replacing the β5-β6 loop in OXA-48 with that of OXA-18 also alters the measured carbapenemase activity (lower kcat values). Site-directed mutagenesis studies of OXA-48 variants indicate that residue 214 (arginine in the wildtype OXA-48) is essential for efficient carbapenem hydrolysis. In recent years, structural studies have yielded a variety of crystal structures of OXA-48 in complex with carbapenems, which shed new light on the acylenzyme (AC) intermediate state. Intriguingly, although the β5-β6 loop is suggested to influence carbapenem activity, the only interaction observed between the substrate and residues within this loop (Thr213-Lys218) is a water-mediated contact between the imipenem 6α-hydroxyethyl hydroxyl and Thr213. Furthermore, bound carbapenem Figure . Crystal structures of OXA-48 complexed with carbapenems. Acylenzyme structures of OXA-48 with imipenem (PDB ID 6P97, green sticks) and meropenem (PDB ID 6P98, light pink sticks) show a highly similar binding pose for both substrates, where main differences lie in the orientation of carbapenem C2 "tail" group. The Ω-loop is highlighted in orange, the β5-β6-loop in yellow, and relevant active site interactions with dashed black lines. The carbapenem pyrroline ring is modelled as the Δ2-tautomer in both structures.