Thesis Cork Institute of Technology, 2019.

Bibliography: (pages 290-322).

Antibiotic resistance is becoming a serious public health concern. Infections that some decades ago could be treated with antibiotics now sometimes do not respond to traditional treatment, causing higher mortality and economic losses. An alternative to the use of antibiotics are bacteria's natural predators, bacteriophages (or phages), and specifically their lytic enzymes. These proteins are produced by phages to degrade bacterial peptidoglycan to inject their genetic material into the bacteria (virion-associated peptidoglycan hydrolases) or to release their progeny once the infection is finished (endolysins). They can be applied exogenously to lyse Gram-positive bacteria or be genetically engineered to lyse gram-negative bacteria. Phages also code for proteins that allow them to reach their receptors (depolymerases) and recognise them (receptor binding proteins or fibres). These receptor-binding proteins can be used to detect and to identify pathogenic bacteria. More knowledge on the structure of these proteins is necessary to understand their mechanisms of action and to design chimeric or active-site mutant encolysins able to lyse different pathogenic bacteria. In this work, the structure of the N-acetylmuramidases from two bacteriophages (pseudomonas bactgeriophage vB_PaeM_KTN6 and Erwinia jumbo bacteriophage vB_EamM-Y3) have been determined by X-ray crystallography and catalytic mechanisms have been proposed. Although both proteins are inverted glycoside hydrolases with similar catalytic mechanisms, they present large morphological differences, illustrating the different ways phages solve similar biological problems. A third structure, the Cysteine-Histidine Aminopeptidase/Hydrolase (CHAP) domain fromt he endolysin of staphylococcal bateriophage K, was determined in the presence of triglycine, an analogue of its natural substrate, revealing a potential secondary ligand binding site. Additional endolysins and fibres targeting both Gram-positive and Gram-negative bacteria have been purified and crystallized, increasing the knowledge of these proteins. In the cases where their structure could not be obtained, in-silico models were generated to explore the mechanisms of these enzymes - (author's abstract).