PMID:7925454

Citation	Caldentey, J, Hänninen, AL and Bamford, DH (1994) Gene XV of bacteriophage PRD1 encodes a lytic enzyme with muramidase activity. Eur. J. Biochem. 225:341-6
Abstract	Bacteriophage PRD1 is a lipid-containing virus that infects a variety of Gram-negative bacteria, including Escherichia coli. The phage lyses its host by virtue of a virally-encoded lytic enzyme, the synthesis of which has been assigned to gene XV on the basis of complementation analysis and experiments with mutant phages. We report here the cloning of gene XV into an expression plasmid and the purification of its product, protein P15, to near homogeneity. The purified protein P15, identified by N-terminal sequence analysis, showed a strong lytic activity against chloroform-treated Gram-negative cells. No activity against Gram-positive bacterial species could be detected. The pH optimum of the enzyme was between 7.0-8.0. Protein P15 was readily inactivated at temperatures above 4 degrees C, as well as by increasing the ionic strength of the buffers. The analysis of cell wall digests indicated that P15 is a glycosidase that cleaves the beta (1-4) linkage between N-acetylmuramic acid and N-acetylglucosamine, thus displaying muramidase activity.
Links	PubMed
Keywords	Amino Acid Sequence; Bacteriophages/enzymology; Bacteriophages/genetics; Base Sequence; Cloning, Molecular; DNA Primers; Escherichia coli/virology; Gene Expression; Genes, Viral; Genetic Complementation Test; Gram-Negative Bacteria/virology; Kinetics; Molecular Sequence Data; Muramidase/biosynthesis; Muramidase/genetics; Muramidase/metabolism; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Species Specificity; Viral Proteins/biosynthesis; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPPRD:VLYS		GO:0003796: lysozyme activity	ECO:0000314:		F		Figure 2	complete CACAO 5755
BPPRD:VLYS		GO:0009268: response to pH	ECO:0000314:		P		Figure 2	complete CACAO 5756
BPPRD:VLYS		GO:0009408: response to heat	ECO:0000314:		P		Figure 2	complete CACAO 5757
BPPRD:VLYS		GO:0044277: cell wall disassembly	ECO:0000314:		P		Figure 3	complete CACAO 5758
BPPRD:VLYS	involved_in	GO:0044277: cell wall disassembly	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
BPPRD:VLYS	enables	GO:0003796: lysozyme activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
BPPRD:ENLYS		GO:0033922: peptidoglycan beta-N-acetylmuramidase activity	ECO:0000314:		F		In figure 3, samples of cell walls were combined with the enzyme P15, hydrolyzed with HCl, and chromatographed on a column. Eluted fractions were then tested for radioactivity. Only the sample with [3H]muramicitol was seen only in the tubes with P15 and cell walls, not in any of the control. This meant that P15 bound to [3H]muramicitol and hydrolyzed it, which would lead to cell death. It had previously been discovered that adding p15 to a tube of E. coli would decrease the turbidity of the tube by killing the cells. Figure 3 was just to show the nature of the cells' death.	complete CACAO 13267
BPPRD:ENLYS	involved_in	GO:0044277: cell wall disassembly	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
BPPRD:ENLYS	enables	GO:0003796: lysozyme activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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