Characterization of Vibrio cholerae Neuraminidase by a Novel Mechanism-Based Fluorescent Labeling Reagent

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid residues from higher-order gangliosides to an unmasked GM1, the essential receptor for cholera toxin. Here we report that a novel mechanism-based fluorescent labeling reagent, 5-acetamido-2-(4-N-5-dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosonic acid (1), becomes a unique irreversible inhibitor of VCNA. Characterization of an inactivated VCNA by MALDI-TOF/TOFMS analysis revealed that the Asp-576 and Arg-577 residues, which are located within the 576DRFF579 sequence, were specifically labeled with this suicide-type fluorescent substrate. Neither Asp-576 nor Arg-577 has ever been known to contribute to a specific residue in the rigid and highly conserved active site of VCNA investigated by crystallographic analysis, suggesting that a flexible β-turn structure containing this sequence may have a crucial role in the dynamic nature of substrate recognition and catalytic action by VCNA.