title: Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1.
abstract: Metallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.
authors.affiliation: The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Susann
authors.initials: S
authors.lastname: Skagseth
authors.affiliation: The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Trine Josefine
authors.initials: TJ
authors.lastname: Carlsen
authors.affiliation: The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, Tromsø, Norway Centre for Applied Biotechnology, Uni Research AS, Bergen, Norway.
authors.forename: Gro Elin Kjæreng
authors.initials: GE
authors.lastname: Bjerga
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Clifton, Bristol, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Spencer
authors.affiliation: Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway (UNN), Tromsø, Norway Department of Pharmacy, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Ørjan
authors.initials: Ø
authors.lastname: Samuelsen
authors.affiliation: The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, Tromsø, Norway hanna-kirsti.leiros@uit.no.
authors.forename: Hanna-Kirsti S
authors.initials: HK
authors.lastname: Leiros
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000667:Ampicillin / Q000378:metabolism; D020134:Catalytic Domain; D002442:Ceftazidime / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006868:Hydrolysis; D015378:Imipenem / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D015378:Imipenem; D000667:Ampicillin; D002442:Ceftazidime; D001618:beta-Lactamases; C495217:beta-lactamase GIM-1, Pseudomonas aeruginosa