Abstract :
[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.
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