Proteolytic processing and oligomerization of bacteriophage-derived endosialidases.

Bacteriophages infecting the neuroinvasive pathogen Escherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host. Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages phi K1E, phi K1F, and 63D, respectively. The cloned sequences share a highly conserved catalytic domain but differ in the length of the N- and C-terminal parts. Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF. Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation. By reinvestigation of the respective gene locus in the phi K1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein. Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone. Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain. Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the C-terminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF.