title: Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.
abstract: Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains "atypical" second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Lisandro J
authors.initials: LJ
authors.lastname: González
authors.affiliation: Instituto de Química Rosario (IQUIR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Diego M
authors.initials: DM
authors.lastname: Moreno
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center and Departments of Medicine, Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio, United States of America.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D004795:Enzyme Stability; D009154:Mutation; D019897:Periplasm / Q000201:enzymology* / Q000235:genetics; D011552:Pseudomonas Infections / Q000201:enzymology / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D013379:Substrate Specificity; D018440:beta-Lactam Resistance / Q000502:physiology; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C476155:beta-lactamase SPM-1, Pseudomonas aeruginosa; D001618:beta-Lactamases