Affiliation:
1. Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India
Abstract
ABSTRACT
New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of
Enterobacteriaceae
as a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned
bla
NDM-1
from an
Enterobacter cloacae
clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the
K
m
was reduced, while the
k
cat
was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the Δ
G
D
o
and
T
m
of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.
Funder
Department of Biotechnology, Ministry of Science and Technology
Indian Council of Medical Research
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Cited by
35 articles.
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