Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35

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, andkca...

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Veröffentlicht in:	Antimicrobial agents and chemotherapy 2016-04, Vol.60 (4), p.2366-2372
Hauptverfasser:	Marcoccia, Francesca, Bottoni, Carlo, Sabatini, Alessia, Colapietro, Martina, Mercuri, Paola Sandra, Galleni, Moreno, Kerff, Frédéric, Matagne, André, Celenza, Giuseppe, Amicosante, Gianfranco, Perilli, Mariagrazia
Format:	Artikel
Sprache:	eng
Schlagworte:	Amino Acid Substitution Anti-Bacterial Agents Anti-Bacterial Agents - chemistry Anti-Bacterial Agents - pharmacology beta-Lactam Resistance beta-Lactam Resistance - genetics beta-lactamase beta-Lactamases beta-Lactamases - chemistry beta-Lactamases - genetics beta-Lactamases - metabolism Biocatalysis Biochemistry, biophysics & molecular biology Biochimie, biophysique & biologie moléculaire Catalytic Domain Cephalosporins Cephalosporins - chemistry Cephalosporins - pharmacology circular dichroism Cloning, Molecular enzyme kinetics Escherichia coli Escherichia coli - drug effects Escherichia coli - enzymology Escherichia coli - genetics Gene Expression Isoleucine Isoleucine - chemistry Isoleucine - metabolism Kinetics Life sciences Mechanisms of Resistance metalloenzyme Models, Molecular Mutation NMM-1 Protein Structure, Secondary Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Sciences du vivant Serine - chemistry Serine - metabolism Threonine - chemistry Threonine - metabolism
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Zusammenfassung:	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.
ISSN:	0066-4804 1098-6596 1098-6596
DOI:	10.1128/AAC.00531-15