ИСТИНА

The microbial resistance to antibiotics is a genuine global threat [Antimicrobial Resistance Global Report on surveillance 2014]. Bacterial enzymes beta-lactamases represent the main mechanism by which pathogenic bacteria become resistant to beta-lactam antibiotics, the most widely used class of antibacterial drugs. Appearance and spread of these enzymes represents a constant challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. The number of described unique β-lactamases obtained from clinical isolates is estimated to be at least 1,300. The evolutionary ability of bacteria to adapt to their environment is the driving force for the increasing rates of resistance. The present work includes the creation of an integrated approach to study of the mutual influence of point mutations on the local structure, activity and stability of TEM-type beta-lactamases, extending substrate specificity range and analysis of mutations on the protein globule surface, search the surface regions of the molecule of the enzyme affecting the catalytic activity and stability in conjunction with active site mutation. At present, the data bank of amino acid sequences of β-lactamases (http://www.lahey.org/Studies/temtable.asp/; last accessed August 2016) describes 205 unique enzymes of TEM group. The functional role is studied for no more than 10-12% of mutations of TEM-type β-lactamases. Based on their influence on the catalytic properties they are divided into two groups: key mutations and so called secondary or related ones. It was revealed that key mutations are of two types: mutations that extend the range of substrate specificity (104, 164, 238 and 240), and mutations leading to resistance to inhibitors of β-lactam nature (clavulanic acid, sulbactam, tazobactam) (69, 130, 244 , 275 and 276). Related mutations are located generally away from the catalytic center and enzyme binding site. The most studied is the substitution at position 182, for which it was shown a stabilizing effect in combination with key functional mutations G238S and M69I. The accumulated literature data suggest that it is important not only the identification of the role of different mutations, but study of the mutual influence of the mutations in various combinations. The aim of this work was to identify the role of Q39K mutation. This mutation was the first described natural mutation in the β-lactamase. This replacement relates to the most common and described for all phenotypes of β-lactamases including ESBL (phenotype 2be) and IR (phenotype 2br). We have studied recombinant forms of β-lactamases TEM-1 with single substitutions Q39K, M69V, E104K, R164S, and their combinations. The kinetic properties of the obtained mutants and kinetic thermal stability were investigated. Destabilizing effect of Q39K replacement has been identified in conjunction with key substitutions at positions 69, 104, 164. Analysis of the TEM-1 and its tertiary structure of the mutants by computer modeling and molecular dynamics approaches allowed to suggest an explanation for the changes observed. Study of the mutual influence of a combination of such mutations on the structure and stability of enzymes is of great interest due to its fundamental and practical importance. It will provide new knowledge on the nature of β-lactamases polymorphism, as well as to find the new ways to combat the resistance of microorganisms to antibiotics. We acknowledge the support of Russian Science Foundation (project no. 15-14-00014).