Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. the misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding β-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used β-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent β-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. previous studies have demonstrated that their β-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of diverse substrates by these enzymes. Antibiotic resistance is a rising socioeconomic problem due to the lack of industrial antibiotic development over the last two decades 1,2. Although new antibiotic substances are developed continuously by academic groups, very few are transferred into industrial production and clinical application. Moreover, there is a lack of new (bio) chemical strategies to overcome antibiotic resistance, and although many of the newly developed antibiotic compounds may display novel structural features they mostly act upon the same cellular mechanisms (i.e. inhibition of cell wall biosynthesis, blocking ribosome function or cell replication). Therefore, there is an imminent global threat that available antibiotic substances may not be effective against ever faster evolving microbial pathogens; the most prominent among these pathogens are listed by the WHO and categorised in class 1-3 threat levels 3,4. To meet the societal challenges exerted by these microbial pathogens, new mechanisms to combat antibiotic

β-Lactams are the most widely employed antibiotics in clinical settings due to their broad efficacy and low toxicity. However, since their first use in the 1940s, resistance to β-lactams has proliferated to the point where multi-drug resistant organisms are now one of the greatest threats to global human health. Many bacteria use β-lactamases to inactivate this class of antibiotics via hydrolysis. Although nucleophilic serine-β-lactamases have long been clinically important, most broad-spectrum β-lactamases employ one or two metal ions (likely Zn2+) in catalysis. To date, potent and clinically useful inhibitors of these metallo-β-lactamases (MBLs) have not been available, exacerbating their negative impact on healthcare. MBLs are categorised into three subgroups: B1, B2, and B3 MBLs, depending on their sequence similarities, active site structures, interactions with metal ions, and substrate preferences. The majority of MBLs associated with the spread of antibiotic resistance belong to the B1 subgroup. Most characterized B3 MBLs have been discovered in environmental bacteria, but they are increasingly identified in clinical samples. B3-type MBLs display greater diversity in their active sites than other MBLs. Furthermore, at least one of the known B3-type MBLs is inhibited by the serine-β-lactamase inhibitor clavulanic acid, an observation that may promote the design of derivatives active against a broader range of MBLs. In this Mini Review, recent advances in structure-function relationships of B3-type MBLs will be discussed, with a view to inspiring inhibitor development to combat the growing spread of β-lactam resistance.

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