The enzymes responsible for peptidoglycan (PG) metabolism were examined. Our focus was on autolysin E (AtlE) - a N-acetylglucosaminidase responsible for degradation of PG, and Mur enzymes, responsible for synthesis of PG. The thesis represents the following contributions to the science: detailed information about binding sites and ligands of the PG metabolising enzymes, classification models for the prediction of affinity with Mur enzymes and novel designed fragments with affinity toward AtlE. Such contributions represent a step forward in the development of inhibitors of PG metabolising enzymes.
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COBISS.SI-ID: 300519680The bacteria are surrounded by the cell wall that provides them with mechanical protection. It consists out of a peptidoglycan scaffold, built from polysaccharide strands cross-linked by peptides. Autolysins are enzymes that remodel the cell wall during growth and division. Among them are N-acetylglucosaminidases that cleave the glycosidic bonds between NAG and NAM monosaccharide residues. Here we focused on N-acetylglucosaminidases of the Staphylococcus aureus, a pathogenic microorganism that represents a major public health burden. We have identified four peptidoglycan N-acetylglucosaminidases that are highly conserved in S. aureus, SagB, AtlA-gl, AtlE and ScaH-gl. Crystal structures have revealed that the enzymes have highly similar secondary structure organization and markedly different tertiary structure. After analysis and comparison of their substrate binding sites we proposed that for the enzymatic reaction to proceed a substantial conformational change needs to take place. This leads to closing of the enzyme. Molecular dynamics simulations have confirmed that closed conformations of these enzymes are stable, but only when the substrate is bound. This indicates that the conformational change occurs with binding of the substrate. Study of the catalytic mechanism of AtlE has shown that for of the reaction only one catalytic amino acid is necessary. As the energetically favorable path water-assisted catalysis was established. Additionally, we proposed the transition state of the reaction is represented by oxocarbenium ion. In addition, we have designed the first compounds that AtlE binds. Compounds from the structural class of (phenylureido)piperidinyl benzamides were found to exhibit promising binding affinity in the micromolar range. The results we present here are a basis for further drug target validation of S. aureus N-acetlyglucosaminidases as drug targets.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 297419520