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-alpha-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 (576)DRFF(579) 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 beta-turn structure containing this sequence may have a crucial role in the dynamic nature of substrate recognition and catalytic action by VCNA.
Hinou
Hiroshi
H
Glycochemosynthesis Team, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan.
Kurogochi
Masaki
M
Shimizu
Hiroki
H
Nishimura
Shin-Ichiro
S
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012794:Sialic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014734:Vibrio cholerae / Q000201:enzymology*
C505577:5-acetamido-2-(4-N-5-dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-3,5-dideoxygalacto-2-nonulopyranosonic acid; D005456:Fluorescent Dyes; D012794:Sialic Acids; D001224:Aspartic Acid; D001120:Arginine; D009439:Neuraminidase