Pseudomonas aeruginosa Cryptic Prophage Endolysin Is a Highly Active Muramidase
Per Kristian Thorén Edvardsen1, Andrea Nikoline Englund1, Åsmund Kjendseth Ro̷hr1
1Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås 1432, Norway.
Abstract:
Endolysins are phage-encoded enzymes that cleave the peptidoglycan of host bacteria. These enzymes have gained considerable attention due to their ability to cause cell lysis, making them candidates as antibacterial agents. Most Pseudomonas aeruginosa genomes, including the common laboratory strains PAO1 and UCBPP-PA14, contain a cryptic prophage encoding a glycoside hydrolase family 19 endolysin (named PaGH19Lys in the present study). Family 19 glycoside hydrolases are known to target peptidoglycan and chitin-type substrates. PaGH19Lys was not active toward chitin but exhibited activity toward chloroform-treated Gram-negative bacteria, displaying ∼10,000-fold higher activity than hen egg white lysozyme. Analysis of products derived from PaGH19Lys activity toward purified P. aeruginosa peptidoglycan showed that the enzyme catalyzed hydrolysis of the β-1,4 linkage between N-acetylmuramic acid and N-acetyl-d-glucosamine, classifying the enzyme as a muramidase. Finally, the crystal structure of PaGH19Lys was determined and solved to 1.8 Å resolution. The structure of the enzyme showed a globular α-helical fold possessing a deep but relatively open catalytic cleft.
Insights
Phage endolysins, like PaGH19Lys from Pseudomonas aeruginosa, show potent antibacterial activity by degrading bacterial cell walls. This muramidase enzyme offers a promising new avenue for developing novel antibacterial agents.
Area of Science:
- Microbiology
- Enzymology
- Structural Biology
Background:
- Endolysins are phage-derived enzymes targeting bacterial peptidoglycan.
- Pseudomonas aeruginosa genomes often encode cryptic prophages with potential antibacterial enzymes.
- Glycoside hydrolase family 19 enzymes are known to interact with peptidoglycan and chitin.
Purpose of the Study:
- To characterize the Pseudomonas aeruginosa glycoside hydrolase family 19 endolysin (PaGH19Lys).
- To investigate the enzymatic activity and substrate specificity of PaGH19Lys.
- To determine the crystal structure of PaGH19Lys.
Main Methods:
- Enzyme activity assays using Gram-negative bacteria and purified peptidoglycan.
- Product analysis of enzymatic hydrolysis.
- X-ray crystallography to determine enzyme structure.
Main Results:
- PaGH19Lys demonstrated potent lytic activity against Gram-negative bacteria, significantly exceeding that of hen egg white lysozyme.
- The enzyme specifically hydrolyzed the β-1,4 linkage in Pseudomonas aeruginosa peptidoglycan, acting as a muramidase.
- The crystal structure revealed a globular α-helical fold with an accessible catalytic cleft.
Conclusions:
- PaGH19Lys is a potent muramidase with significant antibacterial potential.
- Its specific peptidoglycan cleavage mechanism highlights its promise as a therapeutic agent.
- Structural insights provide a basis for further enzyme engineering.
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