Abstract
BackgroundVibrio 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.MethodsTwo 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.ResultsSubstitution 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.ConclusionsChemical 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.General SignificanceIdentification 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.
Highlights
Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin biomaterials, which are abundantly available in the aquatic environment, as its sole source of energy
Identification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437
glycoside hydrolase family 3 (GH3) GlcNAcases catalyse the hydrolytic reaction through a standard ‘retaining’ mechanism involving a covalent glycosyl-enzyme intermediate [14,15], while GH20 and GH84 GlcNAcases hydrolyse chitooligosaccharides through a ‘substrate-assisted’ mechanism involving the transient formation of an oxazolinium ion intermediate [16,17,18]
Summary
Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin biomaterials, which are abundantly available in the aquatic environment, as its sole source of energy. A copper-dependent enzyme, attacks recalcitrant chitin polysaccharides [4,5], while endochitinases hydrolyse long chitin chains to chitin oligosaccharides, which are transported through the bacterial cell wall by chitoporin or ChiP [6,7,8]. In the periplasm, these chitin fragments are degraded by exo β-N-acetylglucosaminidases (GlcNAcases) and the resultant GlcNAc monomers are transported through the inner membrane by the GlcNAc-PTS transporter and metabolized in the cytoplasm, acting as sources of carbon and nitrogen [9]. 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
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