Exploring the role of L209 residue in the active site of NDM-1 a metallo-β-lactamase

• Published in: PloS one Vol. 13; no. 1; p. e0189686
• Main Authors: Marcoccia, Francesca, Leiros, Hanna-Kirsti S, Aschi, Massimiliano, Amicosante, Gianfranco, Perilli, Mariagrazia
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.01.2018; Public Library of Science (PLoS)
• Subjects: Antibiotics; Antimicrobial agents; Bacteria; Benzylpenicillin; Biology and Life Sciences; Carbapenems; Catalysis; Cephalosporins; Chemistry: 440; Cloning; Destabilization; Drug development; Drug resistance; E coli; Enzymes; Flexibility; Gram-negative bacteria; Kjemi: 440; Leucine; Matematikk og Naturvitenskap: 400; Mathematics and natural science: 400; Medicine and Health Sciences; Metallography; Moisture content; Molecular dynamics; Mutagenesis; Mutation; Phenylalanine; Physical Sciences; Point mutation; Proteins; Research and Analysis Methods; VDP; Water content; Zinc; United States > US; Norway; Italy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0189686

New Delhi Metallo-β-Lactamase (NDM-1) is one of the most recent additions to the β-lactamases family. Since its discovery in 2009, NDM-1 producing Enterobacteriaceae have disseminated globally. With few effective antibiotics against NDM-1 producers, there is an urgent need to design new drug inhibitors through the help of structural and mechanistic information available from mutagenic studies. In our study we focus the attention on the non-catalytic residue Leucine 209 by changing it into a Phenylalanine. The L209F laboratory variant of NDM-1 displays a drastic reduction of catalytic efficiency (due to low kcat values) towards penicillins, cephalosporins and carbapenems. Thermofluor-based assay demonstrated that NDM-1 and L209F are stable to the temperature and the zinc content is the same in both enzymes as demonstrated by experiments with PAR in the presence of GdnHCL. Molecular Dynamics (MDs) simulations, carried out on NDM-1 and L209F both complexed and uncomplexed with Benzylpenicillin indicate that the point mutation produces a significant mechanical destabilization of the enzyme and also an increase of water content. These observations clearly show that the single mutation induces drastic changes in the enzyme properties which can be related to the observed different catalytic behavior.