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. 2006 Apr 1;395(1):31-7.
doi: 10.1042/BJ20051780.

Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum

Edward J Taylor  1 , Nicola L Smith, Johan P Turkenburg, Simone D'Souza, Harry J Gilbert, Gideon J Davies
Affiliations

Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum

Edward J Taylor et al. Biochem J. .

Abstract

The digestion of the plant cell wall requires the concerted action of a diverse repertoire of enzyme activities. An important component of these hydrolase consortia are arabinofuranosidases, which release L-arabinofuranose moieties from a range of plant structural polysaccharides. The anaerobic bacterium Clostridium thermocellum, a highly efficient plant cell wall degrader, possesses a single alpha-L-arabinofuranosidase (EC 3.2.1.55), CtAraf51A, located in GH51 (glycoside hydrolase family 51). The crystal structure of the enzyme has been solved in native form and in 'Michaelis' complexes with both alpha-1,5-linked arabinotriose and alpha-1,3 arabinoxylobiose, both forming a hexamer in the asymmetric unit. Kinetic studies reveal that CtAraf51A, in contrast with well-characterized GH51 enzymes including the Cellvibrio japonicus enzyme [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614], catalyses the hydrolysis of alpha-1,5-linked arabino-oligosaccharides and the alpha-1,3 arabinosyl side chain decorations of xylan with equal efficiency. The paucity of direct hydrogen bonds with the aglycone moiety and the flexible conformation adopted by Trp(178), which stacks against the sugar at the +1 subsite, provide a structural explanation for the plasticity in substrate specificity displayed by the clostridial arabinofuranosidase.

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Figures

Figure 1
Figure 1. The enzymatic breakdown of xylan (A) with diverse substrates used in the characterization of CtAraf51 (B)
Figure 2
Figure 2. The three-dimensional structure of CtAraf51
(A) Divergent (wall-eyed) stereo representation of a monomer of CtAraf51 with the ligand observed in the AX2 complex shown in ball-and-stick representation. The cartoon is ‘colour-ramped’ from the N-terminus (blue) to C-terminus (red). (B) A hexamer of CtAraf51 with each monomer in a different colour.
Figure 3
Figure 3. The active centre of CtAraf51
Observed electron density for the AX2 complex (A) and A3 complex (B) of CtAraf51 with interacting residues shown at the electron density contoured at approx. 1σ. (C) The Figure shows the overlap of the two complexes of CtAraf51 (AX2 grey; A3 green) with the AX complex (yellow) of the G. stearothermophilus enzyme [14] in divergent (wall-eyed) stereo. The protein surface (D) reveals that the specificity site for the arabinoside moiety is at the bottom of a funnel-shaped depression which permits accommodation of the terminal arabinoside derived from different oligosaccharides with almost equal catalytic efficiency (the nucleophile is shown in red liquorice and the Ara-Xyl from the AX2 complex in cyan).
Figure 4
Figure 4. Interactions in the active centre of CtAraf51
Hydrogen bonds <3.2 Å are shown as dotted lines.
See this image and copyright information in PMC

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