title: Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35.
abstract: Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Francesca
authors: F
authors: Marcoccia
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Carlo
authors: C
authors: Bottoni
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Alessia
authors: A
authors: Sabatini
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Martina
authors: M
authors: Colapietro
authors: Laboratoire de Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors: Paola Sandra
authors: PS
authors: Mercuri
authors: Laboratoire de Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors: Moreno
authors: M
authors: Galleni
authors: Cristallographie des Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors: Frédéric
authors: F
authors: Kerff
authors: Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors: André
authors: A
authors: Matagne
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Giuseppe
authors: G
authors: Celenza
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
authors: Gianfranco
authors: G
authors: Amicosante
authors: Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy perilli@univaq.it.
authors: Mariagrazia
authors: M
authors: Perilli
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D055162:Biocatalysis; D020134:Catalytic Domain; D002511:Cephalosporins / Q000737:chemistry* / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007532:Isoleucine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D011994:Recombinant Proteins; D007532:Isoleucine; D013912:Threonine; C054920:loracarbef; D012694:Serine; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1