Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies

Alessandra Piccirilli; Emanuele Criscuolo; Fabrizia Brisdelli; Paola Sandra Mercuri; Sabrina Cherubini; Maria Laura De Sciscio; Mauro Maccarrone; Moreno Galleni; Gianfranco Amicosante; Mariagrazia Perilli

doi:10.3390/molecules26185489

Molecules (Sep 2021)

Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies

Alessandra Piccirilli,
Emanuele Criscuolo,
Fabrizia Brisdelli,
Paola Sandra Mercuri,
Sabrina Cherubini,
Maria Laura De Sciscio,
Mauro Maccarrone,
Moreno Galleni,
Gianfranco Amicosante,
Mariagrazia Perilli

Affiliations

Alessandra Piccirilli: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
Emanuele Criscuolo: Department of Experimental Medicine, Tor Vergata University of Rome, Via Montpellier 1, 00121 Rome, Italy
Fabrizia Brisdelli: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
Paola Sandra Mercuri: Macromolécules Biologiques, Centre d’Ingénierie des Protéines, InBioS, Université de Liège, 4000 Liège, Belgium
Sabrina Cherubini: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
Maria Laura De Sciscio: Department of Medicine, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21, 00128 Rome, Italy
Mauro Maccarrone: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
Moreno Galleni: Macromolécules Biologiques, Centre d’Ingénierie des Protéines, InBioS, Université de Liège, 4000 Liège, Belgium
Gianfranco Amicosante: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
Mariagrazia Perilli: Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy

DOI: https://doi.org/10.3390/molecules26185489
Journal volume & issue: Vol. 26, no. 18
p. 5489

Abstract

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Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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