The X-ray structure of Aspergillus aculeatus polygalacturonase and a modeled structure of the polygalacturonase-octagalacturonate complex

Polygalacturonases hydrolyze the alpha-(1-4) glycosidic bonds of de-esterif ied pectate in the smooth region of the plant cell wall. Crystal structures of polygalacturonase from Aspergillus aculeatus were determined at pH 4.5 and 8.5 both to 2.0 Angstrom resolution. A. aculeatus polygalacturonase is a glycoprotein with one N and ten O-glycosylation sites and folds into a ri ght-handed parallel beta -helix. The structures of the three independent mo lecules are essentially the same, showing no dependency on pH or crystal pa cking, and are very similar to that of Aspergillus niger polygalacturonase. However, the structures of the long T1 loop containing a catalytic tyrosin e residue are significantly different in the two proteins. A three-dimensio nal model showing the substrate binding mode for a family 28 hydrolase was obtained by a combined approach of flexible docking, molecular dynamics sim ulations, and energy minimization. The octagalacturonate substrate was mode led as an unbent irregular helix with the -1 ring in a half-chair (H-4(3)) form that approaches the transition state conformation. A comparative model ing of the three polygalacturonases with known structure shows that six sub sites ranging from -4 to +2 are clearly defined but subsites -5 and +3 may or may not be shaped depending on the nearby amino acid residues. Both dist al. subsites are mostly exposed to the solvent region and have weak binding affinity even if they exist. The complex model provides a clear explanatio n for the functions, either in catalysis or in substrate binding, of all co nserved amino acid residues in the polygalacturonase family of proteins. Mo deling suggests that the role of the conserved Asn157 and Tyr270, which had previously been unidentified, may be in transition state stabilization. In A. niger polygalacturonase, the long T1 loop may have to undergo conformat ional change upon binding of the substrate to bring the tyrosine residue cl ose to subsite -1. (C) 2001 Academic Press.