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

Elina Jakobsson , Anne Jokilammi , Juha Aalto , Pauli Ollikka , Jukka V Lehtonen , Harri Hirvonen , Jukka Finne (1)

1 Medical Biochemistry

Endosialidase is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polysialic acid. Polysialic acid is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the neural cell adhesion molecule NCAM. We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A phage and one of the PK1E phage which display lost or residual enzyme activity but retain the binding activity to polysialic acid. 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 polysialic acid binding and cleaving activities of endosialidase and for the evolvement of host range mutants of E. coli K1 bacteriophages.

Mots clés: Life Sciences

Elina Jakobsson, Anne Jokilammi, Juha Aalto, Pauli Ollikka, Jukka V Lehtonen, et al.. 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. Biochemical Journal, 2007, 405 (3), pp.465-472. ⟨10.1042/BJ20070177⟩. ⟨hal-00478750⟩