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We analyzed the kinetic properties of the metagenomic class B3 β-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-β-lac-tamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB β-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B β-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-β7 and β11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 β-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens. Fil: Rodríguez, María Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Herman, Raphaël. Université de Liège; Bélgica Fil: Ghiglione, Barbara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Kerff, Frédéric. Université de Liège; Bélgica Fil: D'amico González, Gabriela Elena Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Bouillenne, Fabrice. Université de Liège; Bélgica Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Handelsman, Jo. University of Yale; Estados Unidos Fil: Charlier, Paulette. Université de Liège; Bélgica Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Sauvage, Eric. Université de Liège; Bélgica Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.