Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages.
Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.
Jakobsson
Elina
E
Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland.
Jokilammi
Anne
A
Aalto
Juha
J
Ollikka
Pauli
P
Lehtonen
Jukka V
JV
Hirvonen
Harri
H
Finne
Jukka
J
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003090:Coliphages / Q000378:metabolism*; D006224:Cricetinae; D004926:Escherichia coli / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D012794:Sialic Acids / Q000378:metabolism*
D012794:Sialic Acids; C021319:polysialic acid; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase