title: Proteolytic release of the intramolecular chaperone domain confers processivity to endosialidase F.
abstract: Endosialidases (endoNs), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the alpha2,8-linked polysialic acid (polySia) capsules of their hosts. The crystal structure solved for the catalytic domain of endoN from coliphage K1F (endoNF) revealed a functional trimer. Folding of the catalytic trimer is mediated by an intramolecular C-terminal chaperone domain. Release of the chaperone from the folded protein confers kinetic stability to endoNF. In mutant c(S), the replacement of serine 911 by alanine prevents proteolysis and generates an enzyme that varies in activity from wild type. Using soluble polySia as substrate a 3-times higher activity was detected while evaluation with immobilized polySia revealed a 190-fold reduced activity. Importantly, activity of c(S) did not differ from wild type with tetrameric sialic acid, the minimal endoNF substrate. Furthermore, we show that the presence of the chaperone domain in c(S) destabilizes binding to polySia in a similar way as did selective disruption of a polySia binding site in the stalk domain. The improved catalytic efficiency toward soluble polySia observed in these mutants can be explained by higher dissociation and association probabilities, whereas inversely, an impaired processivity was found. The fact that endoNF is a processive enzyme introduces a new molecular basis to explain capsule degradation by bacteriophages, which until now has been regarded as a result of cooperative interaction of tailspike proteins. Moreover, knowing that release of the chaperone domain confers kinetic stability and processivity, conservation of the proteolytic process can be explained by its importance in phage evolution.
authors.affiliation: Institut für Zelluläre Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Schwarzer
authors.forename: Katharina
authors.initials: K
authors.lastname: Stummeyer
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselhorst
authors.forename: Friedrich
authors.initials: F
authors.lastname: Freiberger
authors.forename: Bastian
authors.initials: B
authors.lastname: Rode
authors.forename: Melanie
authors.initials: M
authors.lastname: Grove
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
authors.forename: Martina
authors.initials: M
authors.lastname: Mühlenhoff
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D001665:Binding Sites; D002240:Carbohydrate Sequence; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D018832:Molecular Chaperones; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase; D010447:Peptide Hydrolases; D019158:N-Acetylneuraminic Acid