Crystal structure and characterization of the glycoside hydrolase family 62 {alpha}-L-arabinofuranosidase from Streptomyces coelicolor [Protein Structure and Folding]
January 30th, 2014 by Maehara, T., Fujimoto, Z., Ichinose, H., Michikawa, M., Harazono, K., Kaneko, S.
α-L-Arabinofuranosidase belongs to the glycoside hydrolase family 62 (GH62) hydrolyze 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 from Streptomyces coelicolor, which comprises a carbohydrate binding module family 13 domain at its N-terminus and a catalytic domain at its C-terminus. The catalytic domain was a five-bladed β-propeller comprising 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 catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp202 and Glu361 residues were catalytic residues, and Trp270, Tyr461, and Asn462 residues were involved in the substrate binding site for discriminating substrate structures. In particular, hydrogen bonding between Asn462 and xylose at the +2 nonreducing end subsite was important for higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.