title: Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site.
abstract: Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.
authors.affiliation: Departamento de Química Biológica and Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Jorgelina
authors.initials: J
authors.lastname: Morán-Barrio
authors.affiliation: Departamento de Química Biológica and Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Laboratory of Molecular & Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: María-Natalia
authors.initials: MN
authors.lastname: Lisa
authors.affiliation: Laboratory of Molecular & Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: Nicole
authors.initials: N
authors.lastname: Larrieux
authors.affiliation: Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina Instituto de Biología Molecular y Celular de Rosario (CONICET-UNR), Ocampo y Esmeralda, Rosario, Argentina.
authors.forename: Salvador I
authors.initials: SI
authors.lastname: Drusin
authors.affiliation: Departamento de Química Biológica and Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Alejandro M
authors.initials: AM
authors.lastname: Viale
authors.affiliation: Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina Instituto de Química de Rosario (IQUIR, CONICET-UNR), Rosario, Santa Fe, Argentina.
authors.forename: Diego M
authors.initials: DM
authors.lastname: Moreno
authors.affiliation: Laboratory of Molecular & Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay Département de Biologie Structurale et Chimie, Institut Pasteur, Paris, France vila@ibr-conicet.gov.ar alebus@pasteur.edu.uy.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Buschiazzo
authors.affiliation: Departamento de Química Biológica and Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina vila@ibr-conicet.gov.ar alebus@pasteur.edu.uy.
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D015780:Carbapenems / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002413:Cations, Divalent; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D041965:Flavobacteriaceae / Q000737:chemistry / Q000201:enzymology*; D015870:Gene Expression; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry / Q000201:enzymology; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D000072417:Protein Domains; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002413:Cations, Divalent; D002511:Cephalosporins; D010406:Penicillins; D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D001618:beta-Lactamases; D015032:Zinc