ИСТИНА

Bacterial resistance towards antibiotics has become a global problem in both the medical and agricultural fields, and resistant bacterial strains present an increasing threat to human and animal. Increase in the number of infectious diseases caused by pathogenic bacteria is an acute problem for the whole world, both developed and developing countries. It is necessary to note the significance of the spread of nosocomial infections, often caused by resistant pathogens to antibiotics, which affects primarily people with weakened immune systems: children, the elderly, pregnant women, patients after surgery. By present, resistance mechanisms have been identified and reported for all known antibiotics currently available for clinical use. Currently, beta-lactams are the most commonly used antibiotics. The presence of "chemical core" - the beta-lactam ring is the peculiarity of their chemical structure, and presence of such cyclic amide explains its high reactivity. By now, several mechanisms of bacterial resistance were identified. The most common mechanism of resistance towards beta-lactams is the synthesis of the bacterial beta-lactamases, which hydrolyze beta-lactam ring of the antibiotics. Due to the localization of beta-lactamase encoding genes on mobile genetic elements they spread fast enough, and this escalates additionally the problem of antibiotic resistance to beta-lactam antibiotics. These enzymes are evolving at high rate, amenable to mutations and have noticeable structural variability. The variability of beta-lactamases is a consequence of general biological mechanism of adaptation of microorganisms to antibiotics. A family of TEM-type beta-lactamases involves currently 205 includes enzymes, which are mutant forms of beta-lactamase TEM-1. To date, mutations were described in 92 positions of amino acid chain. Despite intensive research of beta-lactamases, the role of no more than 10-12% of mutations was established. Almost all of them belong to the so-called "key" mutations. They are divided into two groups: mutations which expand the substrate profile and the mutations responsible for the resistance to inhibitors of beta-lactam structure. In the addition to functional key mutations, there are the mutations of residues located far from the catalytic center of the enzyme, so called "secondary" mutations. Among the variety of secondary mutations, the stabilizing effect of M182T replacement was found recently. The aim of our study was to examine the effect of combinations of secondary mutation Q39K with key mutations M69V, E104K, R164S on activity and kinetic stability of the enzymes. Computer modeling allowed the prediction of a chain of hydrogen bonds from glutamine 39 to area of enzyme active site and probable change of protein thermal stability when the replacement of lysine occurs. Study of different recombinant mutant forms of TEM type beta-lactamases containing single and double combination of mutations was performed. It revealed the destabilizing role of the substitution Q39K in a combination with key mutations. The refolding of mutant forms of beta-lactamase TEM-1 was shown to be complex, consisting of several stages: an initial fast phase and the final slow. The last stage of enzyme refolding is assumed to be connected with cis – trans isomerization of Е166 – P167. Only cis –isomer is catalytically active, providing the correct orientation of E166. Our study underlines the relevance of a systematic comprehensive study of the effect of secondary or associated mutations in combination with the key drug resistant mutation on the structure and properties of beta-lactamases. The work was supported by the RSF (Project No. 15-14-00014).