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

SUBMITTER: Meekrathok P 

PROVIDER: S-EPMC4752478 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature