title: Probing the Catalytic Mechanism of Vibrio harveyi GH20 β-N-Acetylglucosaminidase by Chemical Rescue.
abstract: BACKGROUND
  Vibrio harveyi GH20 β-N-acetylglucosaminidase (VhGlcNAcase) is a chitinolytic enzyme responsible for the successive degradation of chitin fragments to GlcNAc monomers, activating the onset of the chitin catabolic cascade in marine Vibrios.
  
  
  METHODS
  Two invariant acidic pairs (Asp303-Asp304 and Asp437-Glu438) of VhGlcNAcase were mutated using a site-directed mutagenesis strategy. The effects of these mutations were examined and the catalytic roles of these active-site residues were elucidated using a chemical rescue approach. Enhancement of the enzymic activity of the VhGlcNAcase mutants was evaluated by a colorimetric assay using pNP-GlcNAc as substrate.
  
  
  RESULTS
  Substitution of Asp303, Asp304, Asp437 or Glu438 with Ala/Asn/Gln produced a dramatic loss of the GlcNAcase activity. However, the activity of the inactive D437A mutant was recovered in the presence of sodium formate. Our kinetic data suggest that formate ion plays a nucleophilic role by mimicking the β-COO-side chain of Asp437, thereby stabilizing the reaction intermediate during both the glycosylation and the deglycosylation steps.
  
  
  CONCLUSIONS
  Chemical rescue of the inactive D437A mutant of VhGlcNAcase by an added nucleophile helped to identify Asp437 as the catalytic nucleophile/base, and hence its acidic partner Glu438 as the catalytic proton donor/acceptor.
  
  
  CONCLUSIONS
  Identification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437-Glu438 for catalysis. The results suggest the mechanistic basis of the participation of β-N-acetylglucosaminidase in the chitin catabolic pathway of marine Vibrios.
authors: Biochemistry-Electrochemistry Research Unit and School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
authors: Piyanat
authors: P
authors: Meekrathok
authors: Biochemistry-Electrochemistry Research Unit and School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.|Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
authors: Wipa
authors: W
authors: Suginta
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D005561:Formates / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity; D014733:Vibrio / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D005561:Formates; C030544:formic acid; D000118:Acetylglucosaminidase