Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 β-N-acetylglucosaminidase from the marine bacteriumVibrio harveyi

Abstract

Vibrio harveyi -N-acetylglucosaminidase (VhGlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å . All crystals were composed of the same type of dimer, with the substrate N,N 0 -diacetylglucosamine (GlcNAc 2 or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.

Characterization of Stackebrandtia nassauensis GH 20 Beta-Hexosaminidase, a Versatile Biocatalyst for Chitobiose Degradation

“…Enzymes in this family operate using a substrate-assisted catalytic mechanism in which the N -acetamido group acts as a nucleophile to displace the anomeric leaving group, which results in a bicyclic oxazoline intermediate. A conserved Asp-Glu amino acid pair act as catalytic acid-base residues to facilitate the departure of the leaving group and polarization of the N -acetamido group [ 2 , 9 , 43 , 44 ]. The mutation of these two residues (D306 and E307) resulted in complete loss of activity when using an N -glycan standard (A2, Table S2 ) as a substrate.…”

NAG‐thiazoline is a potent inhibitor of the Vibrio campbellii GH20 β‐N‐Acetylglucosaminidase

“…Inhibitory effect of NGT on VhGlcNAcase using a chromogenic substrate NAG-thiazoline is an analog of the reaction intermediate for bacterial and human GlcNAcases [24,26] found in family GH20. Its chemical structure closely resembles that of the oxazoline reaction intermediate and NGT, thus potentially acts as a strong inhibitor of VhGlcNAcase (Fig.…”

“…The gene was cloned into a pET23a(+) expression vector and successfully expressed at high levels [17]. VhGlcNAcase is an exo-acting enzyme that degrades chitooligosaccharides from the nonreducing end in a sequential manner, liberating GlcNAc units [17,22,24]. In an effort to gain insight into the biochemistry and biological function of GlcNAc-processing enzymes, small-molecule inhibitors have been developed and a number of inhibitors of both bacterial and human GH20 GlcNAcases have been reported.…”

Structural basis of chitin utilization by a GH20 β-N-acetylglucosaminidase from Vibrio campbellii strain ATCC BAA-1116

“…Preliminary crystallization of VhGlcNAcase, both in the apo form and in complex with N-acetylglucosamine (GlcNAc), was performed as described elsewhere (Meekrathok et al, 2015). Crystallization conditions were screened using sittingdrop vapor diffusion at 20 C with the commercially available screens The JCSG Core Suites I, II, III and IV, The Classics and Classics II Suites, The PACT Suite, The PEGs Suite and The Anions Suite (Qiagen, Hilden, Germany).…”

“…20 mm. These wild-type crystals were subsequently successfully soaked with 10 mM (GlcNAc) 2 in the corresponding mother liquor at 20 C for a period of approximately 30 min, as described previously (Meekrathok et al, 2015). The native and soaked crystals were then transferred into a cryoprotectant solution containing the mother liquor with 2.9 M sodium malonate and 10 mM (GlcNAc) 2 .…”