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

Abstract

Vibrio harveyi β-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'-diacetylglucosamine (GlcNAc₂ or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.

Keywords: GH20 glycoside hydrolase family; Vibrio harveyi; β-N-acetylglucosaminidase.

Figure 1

Purification of VhGlcNAcase expressed from E. coli M15 cells using a HiPrep 16/60 Sephacryl S-200 prepacked gel-filtration column. Chromatography was performed under a low salt concentration, as described in the text. (a) A chromatographic elution profile of wild-type VhGlcNAcase obtained from an ÄKTApurifier system. (b) GlcNAcase-containing fractions obtained from the two peaks were pooled separately and then loaded onto native PAGE followed by Coomassie Blue staining. (c) The same protein fractions were analysed by SDS–PAGE. Lane Std, low-molecular-weight protein markers (labelled in kDa); lane 1, pooled fractions from the void peak; lane 2, pooled fractions from the second peak.

Figure 2

(a) A crystal of wild-type VhGlcNAcase, with dimensions of 400 × 200 × 20 µm, obtained from a hanging-drop vapour-diffusion setup using 0.1 M sodium acetate pH 4.6 containing 1.4 M sodium malonate. (b) A crystal of the D437A mutant (dimensions of 500 × 300 × 50 µm) obtained from a hanging-drop vapour-diffusion setup using 20%(w/v) PEG 3350, 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate. Both crystals were obtained within 3 d of incubation at 293 K.

Figure 3

The electron-density map fitted with a GlcNAc molecule after the wild-type VhGlcNAcase crystals had been soaked with the substrate diNAG for 30 min. The structure is presented at 2.5 Å resolution and is contoured at the 1σ level.

Figure 4

Crystals of the D437A mutant belonging to the tetragonal space group P4₃2₁2. Crystals grew as thin rod-shaped crystals within 14 d of incubation in condition C12 from The Anions Suite (0.1 M MES pH 6.5, 1.2 M sodium malonate) at 293 K.

Figure 5

X-ray diffraction images of (a) wild-type VhGlcNAcase in the absence of diNAG and (b) the inactive D437A mutant with resolutions of 2.4 and 2.6 Å, respectively. The X-ray data were collected on beamline PX-II at the Swiss Light Source, Villigen, Switzerland.