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1.68-Å Crystal Structure of Endopolygalacturonase II fromAspergillus niger and Identification of Active Site Residues by Site-directed Mutagenesis*

Y. van Santen,J. Benen,K. Schröter,K. H. Kalk,S. Armand,J. Visser,B. Dijkstra

Published 1999 in Journal of Biological Chemistry

ABSTRACT

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel β-helix with 10 complete turns. The β-helix is composed of four parallel β-sheets, and has one very small α-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the β-helix. Site-directed mutagenesis of Asp180, Asp201, Asp202, His223, Arg256, and Lys258, which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp180 and Asp202activate the attacking nucleophilic water molecule, while Asp201 protonates the glycosidic oxygen of the scissile bond.

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