Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum

Abstract

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

Keywords: Clostridium thermocellum; Xyn30A; glycoside hydrolase family 30.

Figure 1

(a) SDS–PAGE [13%(w/v)] showing overexpression and purification of Xyn30A. Lane 1, Page Ruler protein marker (labelled in kDa); lane 2, uninduced Xyn30A cells; lane 3, cell-free extract; lane 4, last wash from column; lane 5, purified Xyn30A (∼45 kDa). (b) Crystals of Xyn30A obtained by sitting-drop vapour diffusion. The largest crystals are approximately 200 × 120 × 60 µm in size.

Figure 2

Representative diffraction pattern of a Xyn30A crystal with resolution rings shown as dashed circles. The inset shows an enlargement of the contents of the red box, showing spots at ∼1.2 Å resolution.