title: The essential protein for bacterial flagella formation FlgJ functions as a β-N-acetylglucosaminidase.
abstract: The flagellum is a major virulence factor of motile pathogenic bacteria. This structure requires more than 50 proteins for its biogenesis and function, one of which is FlgJ. Homologs of FlgJ produced by the β- and γ-proteobacteria, such as Salmonella enterica, Vibrio spp., and both Sphingomonas sp. and Pseudomonas spp. are bifunctional, possessing an N-terminal domain responsible for proper rod assembly and a C-terminal domain possessing peptidoglycan lytic activity. Despite the amount of research conducted on FlgJ from these and other bacteria over the past 15 years, no biochemical analysis had been conducted on any FlgJ and consequently confusion exists as to whether the enzyme is a peptidoglycan hydrolase or a lytic transglycosylase. In this study, we present the development of a novel assay for glycoside lytic enzymes and its use to provide the first enzymatic characterization of the lytic domain of FlgJ from S. enterica as the model enzyme. Surprisingly, FlgJ functions as neither a muramidase nor a lytic transglycosylases but rather as a β-N-acetylglucosaminidase. As such, FlgJ represents the first autolysin with this activity to be characterized from a Gram-negative bacterium. At its optimal pH of 4.0, the Michaelis-Menten parameters of K(m) and k(cat) for FlgJ from S. enterica were determined to be 0.64 ± 0.18 mg ml(-1) and 0.13 ± 0.016 s(-1), respectively, using purified PG as substrate. Its catalytic residues were identified as Glu(184) and Glu(223).
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Francesca A
authors.initials: FA
authors.lastname: Herlihey
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Moynihan
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada a.clarke@exec.uoguelph.ca.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Clarke
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D005407:Flagella / Q000378:metabolism*; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism; D016366:Open Reading Frames; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019779:Salmonella enterica / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D000118:Acetylglucosaminidase; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Autolysin; Bacterial Pathogenesis; Cell Wall; Enzyme Catalysis; Flagella; Lytic Transglycosylase; Muramidase; N-Acetylglycosaminidase; Peptidoglycan; Virulence Factor