Computer modeling of the rhamnogalacturonase-"hairy" pectin complex.
The structure of a pectin-bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The "hairy" region of pectin, represented by six repeating stretches of (1-->4)-D-galacturonate-(1-->2)-L-rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X-ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the -1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo-threefold helix with the -1 ring in a 4H3 half-chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic "smooth" region of linear homopolymer of D-(1-->4)-linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from -3 to +7, while polygalacturonase has eight subsites from -5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase.