A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting glycosidase.
Bacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endo-sialidases. This family is unusual because all other previously reported sialidases outside of this family are exo- or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a series of pH values revealed a dependence on a single protonated group of pKa 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1.
Thomas J
TJ
Morley
Lisa M
LM
Willis
Chris
C
Whitfield
Warren W
WW
Wakarchuk
Stephen G
SG
Withers
D000818:Animals; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D016123:Campylobacter jejuni / Q000201:enzymology; D002855:Chromatography, Thin Layer; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology
D006023:Glycoproteins; D006027:Glycosides; D012799:Sialyltransferases; D006026:Glycoside Hydrolases; C035355:endo-N-acetylneuraminidase; C069715:trans-sialidase; D009439:Neuraminidase