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.