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Impact of Residue R65 on the Stabilization of TEM-Type β-Lactamases with Substitution of M182T
Moscow University Chemistry Bulletin ( IF 0.7 ) Pub Date : 2020-07-27 , DOI: 10.3103/s0027131420030049 V. G. Grigorenko , I. P. Andreeva , O. V. Serova , M. Yu. Rubtsova , A. M. Egorov
中文翻译:
残留R65对M182T取代的TEM型β-内酰胺酶稳定性的影响
更新日期:2020-07-27
Moscow University Chemistry Bulletin ( IF 0.7 ) Pub Date : 2020-07-27 , DOI: 10.3103/s0027131420030049 V. G. Grigorenko , I. P. Andreeva , O. V. Serova , M. Yu. Rubtsova , A. M. Egorov
Abstract
For the experimental study of the role of the R65 residue in the stabilization of β-lactamases with the substitution of M182T, predicted by the analysis of the residue interaction networks (RINs), homogeneous preparations of the recombinant TEM-type β-lactamases with substitutions of R65L, M182A, and a combination of R65L and M182T mutations were obtained. The kinetic parameters of these enzymes were determined for penicillin, ceftazidime, cephalothin and CENTA. None of the investigated substitutions changed the substrate specificity of enzymes against β-lactam antibiotics. The substitution R65L leads to a decrease in thermal stability; the substitution of M182A and the combination of substitutions of R65L and M182T improve the thermal stability of β-lactamase in comparison with the wild-type enzyme TEM-1. Using differential scanning calorimetry, it was determined the enthalpy (ΔH, kJ/mol) and the denaturation temperature (Tmp, °C) for β-lactamases TEM-1, TEM(M182A), TEM-135(M182T), and TEM (R65L + M182T), which are, respectively, 554.0 and 50.8, 573.7 and 51.7, 654.4 and 55.9, and 647.4 and 51.9. 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 the R65 residue and the appearance of its new contacts with the residues of the Ω-loop of β-lactamase.中文翻译:
残留R65对M182T取代的TEM型β-内酰胺酶稳定性的影响