The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

Abstract

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a b-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating b-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites -4 and -3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.

A myxobacterial GH19 lysozyme with bacteriolytic activity on both Gram-positive and negative phytopathogens

“…Two proteins, WP_143898266.1 assigned as C25GH19A and WP_161664743.1 assigned as C25GH19B, were annotated as glycoside hydrolase 19 (GH19) family hydrolases. The GH19 family includes chitinases derived from bacteria and plants, and lysozymes or endolysins derived virus Ma-LMM01 (Hosoda et al 2011) and the endolysin (AEX26904.1) derived from Salmonella phage SPN1S (Park et al 2014) is 54% and 31%, respectively. According to the multiple sequence alignment (Fig.…”

Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature

“…Besides, a number of papers [ 20 , 21 ] pointed out that some GH19 proteins are coded by single genes or modular multi-domain lytic gene cassettes in bacteriophages or prophages. A few of these enzymes have been biochemically characterized and classified as endolysins with lysozyme-like properties [ 22 , 23 , 24 , 25 , 26 , 27 ]. Endolysins are produced in the late phases of the bacteriophage’s lytic cycle and attack the peptidoglycan polymer of the host cell wall, thus allowing for the release of the phage progeny.…”

Chitinases as Antibacterial Proteins: A Systematic Review

“…However, it has been shown that some chitinases from diverse sources, besides being able to cleave the O-glycosidic bonds in chitin chains, also have lysozyme activity. 25,26,27 Hevamine, a GH18 chitinase firstly purified from the latex of Hevea brasiliensis, is probably the better known chitinase with lysozyme activity. 28,29 Most bacterial cells are encased by a cell wall constituted mainly by a mesh-like layer of peptidoglycan (PG), also known as murein, which guarantees cell integrity and shape.…”