Crystal structure and characterization of the glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor

Abstract

α-L-arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-L-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp(202) and Glu(361) were catalytic residues, and Trp(270), Tyr(461), and Asn(462) were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn(462) and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.

Keywords: Crystal Structure; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Mutation; Enzyme Structure; Glycoside Hydrolase Family 62; Streptomyces coelicolor; Substrate Specificity; α -l-Arabinofuranosidase.

FIGURE 1.

Enzymatic properties of wild-type α-l-arabinofuranosidase from S. coelicolor (ScAraf62A). A, optimum pH; B, optimum temperature; C, stability for pH; D, stability for temperature. Symbols used are as follows: black triangle, glycine-HCl buffer; black circle, McIlvaine buffer; black square, Atkins-Pantin buffer.

FIGURE 2.

Structure of α-l-arabinofuranosidase from S. coelicolor (ScAraf62A) catalytic domain. A, stereoview of the ribbon model of the ScAraf62A-l-arabinofuranose complex structure. The bound sugar, catalytically important residues, and disulfide bridge are shown as stick models, and the bound sodium ion is shown as a purple sphere. B, topological diagram of ScAraf62A. 3₁₀-Helices and β-strands are shown as shaded cylinders and filled arrows, respectively.

FIGURE 3.

Stereo view of the α-l-arabinofuranosidase from S. coelicolor (ScAraf62A)-l-arabinose complex catalytic pocket. Yellow stick model, bound Ara; pale red, three catalytically important residues; broken lines, estimated hydrogen bonds; magenta, 2F_o − F_c electron density map of bound Ara (contour level, 1σ). Sugar carbon atoms are numbered.

FIGURE 4.

Bound ligand structures of α-l-arabinofuranosidase from S. coelicolor (ScAraf62A). A, surface model of the ScAraf62A-xylohexaose (X₆) complex catalytic cleft. Gray stick model, bound X₆; pale red, gray stick model, bound Tris molecule; broken lines, estimated hydrogen bonds; magenta, 2F_o − F_c electron density map of bound X₆ (contour level, 1σ). B, superimposed model of l-arabinose (Ara) of ScAraf62A-Ara complex structure (yellow stick model) on the ScAraf62A-X₆ complex structure around the catalytic center. C, superimposed model of the ligand-free structure (light green stick model) on the ScAraf62A-Ara complex structure (yellow-orange-pale red) around the catalytic center. D, schematic representation of the binding model of arabinoxylan to ScAraf62A. Hexagon, xylose; pentagon, Ara.

FIGURE 5.

Active site cleft of α-l-arabinofuranosidase from S. coelicolor (ScAraf62A). Yellow, xylotriose; pink, arabinoheptaose.

FIGURE 6.

Superimposition of α-l-arabinofuranosidase from S. coelicolor (ScAraf62A) and two GH43 AXH catalytic domain structures. A, overall ribbon model. ScAraf62A-xylahexaose (X₆) complex, orange and pink; B. subtilis arabinoxylan α-l-1,3-arabinofuranohydrolase complexed with xylotetraose (PDB code 3C7G) (25), cyan and blue; H. insolens double substituted xylan α-l-1,3-arabinofuranosidase complexed with 3²-α-l-arabinofuranosyl-xylotriose (PDB code 3ZXK) (28), pale green and green. B, close-up view of the three catalytically important residues superimposed on the bound l-arabinose (Ara) molecule in the ScAraf62A-Ara complex. C, close-up view of the catalytic cleft with bound xylooligosaccharides.