title: Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases.
abstract: Escherichia coli phage N4 infection leads to delayed host cell lysis, 3000 particles per infected bacterium and a small plaque phenotype. We show that bacteriophage N4 encodes a murein hydrolase (gp61) that is essential for N4 plaque-forming ability. gp61 has a high level of sequence similarity to hypothetical proteobacterial proteins, and Vibrio harveyi phage VHML ORF 19. Nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) analysis of muropeptides from purified gp61 digestion of E. coli peptidoglycan indicates that gp61 is an N-acetylmuramidase. The EGGY motif present near the N terminus of gp61 and its homologs contains the glutamic acid residue essential for enzymatic activity. These results provide evidence that N4 gp61 and its homologs define a new family of N-acetylmuramidases (pfam05838.4, DUF847, COG3926). In contrast to its homologs, gp61 contains an N-terminal signal sequence. When expressed at levels present during phage infection, gp61 localizes primarily to the cell inner membrane; in contrast, over-expression of recombinant N4 gp61 is sufficient for rapid cell lysis. Overproduction of the recombinant Salmonella typhimurium (STM0016) homolog is sufficient for cell lysis only when fused to the gp61 N-terminal signal sequence. The results of subcellular localization and of mutagenesis of the gp61 N-terminal signal sequence indicate that gp61 must be released from the inner membrane to be catalytically active.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Emina A
authors.initials: EA
authors.lastname: Stojković
authors.forename: Lucia B
authors.initials: LB
authors.lastname: Rothman-Denes
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D025522:Bacteriophage N4 / Q000201:enzymology*; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D012485:Salmonella typhi / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D018698:Glutamic Acid; D004492:Edetic Acid; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase; D009238:N-Acetylmuramoyl-L-alanine Amidase