Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin‐encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity). The protein purified was shown to be an l‐alanoyl‐d‐glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers (25–50 mm) at pH 8.5. The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium/manganese chlorides. It was found that, along with E. coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram‐negative microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis). This endolysin is the first example of an l‐alanoyl‐d‐glutamate peptidase in a virulent phage infecting Gram‐negative bacteria. There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic l‐alanoyl‐d‐glutamate peptidases. The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution.

The 3D-structure solution structure of the Zn ²⁺ -containing form of the bacteriophage T5 EndoT5-Zn ²⁺ is determined by the high-resolution NMR spectroscopy. The structural integrity of the entire molecule is controlled by the Zn ²⁺ binding.

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca²⁺-dependent L-alanyl-D-glutamate peptidase, although the mode of Ca²⁺ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111–130) in Ca²⁺ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca²⁺-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca²⁺ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active “open” conformation. The possible mechanism of Ca²⁺ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca²⁺ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

Identification and cloning of genes as well as biochemical characterization of the gene products were carried out for two novel endolysins of pseudo T-even lytic bacteriophages RB43 and RB49, which represent different myovirus groups of the subfamily Tevenvirinae. Genes RB43ORF159c and RB49р102 were cloned in E. coli cells, and their products were purified to electrophoretic homogeneity with an up to 80 % yield of total activity. In respect to substrate specificity, both enzymes were found to be lytic l-alanoyl-d-glutamate peptidases belonging to the M15 family. The pH optimum functioning of both endolysins was within the range 7.0–9.0, whereas the optimal values of ionic strength were different for the two proteins (25 mM vs 100 mM for the RB43 and RB49 endolysins respectively). Both peptidases were thermally resistant, with the RB43 endolysin being more stable (it restored 81 % of enzyme activity and 96 % of secondary structure after a 10 min heating at 90 °C) than its RB49 counterpart (27 and 77% respectively). The possible origin of genes of lytic l-alanoyl-d-glutamate peptidases of myoviruses as a result of horizontal transfer in the variable parts of genomes between unrelated phages having a common host is discussed.