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S C I E N T I F I C H I G H L I G H T S

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Understanding the metal-dependent oligomerization of PIB-1 and its effect on enzymatic activity

X-ray crystallography has elucidated the metal- dependent structure and function of PIB-1, a b-lactamase from Pseudomonas aeruginosa. This study reveals how metals promote oligomerization and enhance enzymatic activity, providing potential pathways for designing inhibitors targeting metal- regulated b-lactamases to combat antibiotic- resistant bacterial infections.

The rise of antibiotic resistance has been recognized as one of the most significant global challenges for human health. Knowing the mechanisms involved in the acquisition of resistance is critical for developing anti-resistance adjuvants (i.e., b-lactamase inhibitors) and evolution-based strategies to deal with the problem of antibiotic resistance. Antibiotic-inactivating enzymes constitute one of the most relevant mechanisms of resistance and, among them, b-lactamase enzymes stand out as a primary defence [1], being particularly relevant in the case of Gram-negative pathogens. These enzymes inactivate b-lactam antibiotics

by hydrolysing their b-lactam ring, rendering the antibiotic ineffective. Currently, b-lactamases are classified into four groups, according to their functional and structural characteristics. Three of these groups, Groups A, C and D, possess a conserved serine residue in their active sites, while the fourth, Group B, contains zinc ions in its active sites, essential for activity [2]. The zinc-dependence of Group B has highlighted metal ions as critical co-factors in the function of specific b-lactamases.

Several bacteria present chromosomally encoded Class C b-lactamases, which are part of their intrinsic resistome. PIB-1 is a chromosomally encoded b-lactamase from Pseudomonas aeruginosa, initially classified as a potential member of the Class C of b-lactamases based on sequence analysis, yet it does not exhibit the three catalytic conserved motifs typical of this class. Functional analysis showed that PIB-1 is able to degrade carbapenems but not cephalosporins, the typical substrate of Class C b-lactamases. Furthermore, the enzymatic activity of the protein showed a dependence on zinc ions [3], but without the typical signature of the metallo-b-lactamases. Phylogenetic analysis shows that PIB-1 forms an outgroup within the Class C b-lactamases.

To elucidate the mechanistic details of PIB-1’s activity, structural and biochemical studies were carried out, including X-ray diffraction experiments performed at beamline ID29. The resulting crystal structure (Figure 11a) confirmed PIB- 1’s classification within Class C b-lactamases but revealed structural distinctions not apparent through sequence alignment alone. Novel structural elements were identified at the P-, R2- and W-loops, regions that diverge from typical Class C b-lactamase architecture. Crystallographic studies resolved PIB-1 in complex with the carbapenem antibiotic meropenem bound to its catalytic serine (Figure 11b) and in the presence of zinc ions (Figure 11c), suggesting an unusual metal-mediated structural adaptation.

Fig. 11: Structural features of PIB-1. a) Overall structure of the PIB-1 monomer, highlighting key structural elements. b) Electron density map showing the carbapenem antibiotic meropenem bound within the active site of PIB-1. c) Close-up of the zinc- binding site, illustrating the coordination of the zinc ion within the protein structure.