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V. G. Grigorenko, I. P. Andreeva, O. V. Serova, M. Yu. Rubtsova, A. M. Egorov
Impact of residue R65 on the stabilization of TEM-type β-lactamases with substitution M182T
Abstract
For experimental confirmation
the role of residue R65 in the stabilization of TEM type β-lactamases with the M182T
substitution, predicted by the residues interactions networks (RINs) analysis,
homogeneous recombinant TEM type β-lactamases with R65L, M182A
and a combination of R65L and M182T substitutions were obtained. The kinetic
parameters of these enzymes were determined toward penicillin, ceftazidime,
cephalotin, and CENTA. It was shown that all investigated substitutions did not
change the substrate specificity of enzymes against β-lactams. The substitution
of R65L led to a decrease in thermal stability, while the replacement M182A and
the combination of substitutions of R65L and M182T led to an increase in the
thermal stability of β-lactamase compared to TEM-1
β-lactamase.
Using differential scanning calorimetry, the enthalpies (ΔH, kJ/mol) and
denaturation temperatures (Tm, 0C) were determined for β-lactamases TEM-1, TEM
(M182A), TEM-135 (M182T), TEM (R65L + M182T), and the values amounted to 554.0
and 50.8; 573.7 and 51.7; 654.4 and 55.9; 647.4 and 51.9, respectively. The
hypothesis of molecular mechanism, explaining the stabilizing role of M182T
substitution in TEM type β-lactamases, was
supplemented by the effect of changing the conformation of residue R65 and the
establishing of its new contacts with the -loop residues.
Key words:
recombinant TEM type β-lactamases, mutations, thermal
stability, RINs (Residue Interaction Networks).
Copyright (C) Chemistry Dept., Moscow State University, 2002
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