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

[en] 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 -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 -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.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

Piccirilli, Alessandra

Criscuolo, Emanuele

Brisdelli, Fabrizia

Mercuri, Paola ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques

Cherubini, Sabrina

De Scicio, Maria laura

Maccarrone, Mauro

Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques

Amicosante, Gianfranco

Perilli, Mariagrazia

Language :

English

Title :

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

Publication date :

2021

Journal title :

Molecules

Volume :

Pages :

5489

Peer reviewed :

Peer reviewed

Available on ORBi :

since 16 September 2021

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