RHAMNOGALACTURONASE A FROM ASPERGILLUS ACULEATUS. A RIGHT-HANDED [[beta]]-HELIX FOLD. By Thomas Nordahl Petersen and Sine Larsen, Centre for Crystallographic Studies, University of Copenhagen, Denmark

The crystal structure of this plant cell wall degrading enzyme has been determined to 2.0 Å resolution, using the SIRAS method.

Pectin is the major component of a plant cell wall. It consist of a smooth region of homogalacturonan and a 'hairy region' of rhamnogalacturonan. The backbone of rhamnogalacturonan is composed of a chain of alternating rhamnose and galacturonic acid residues [[alpha]]-L-Rha(1-4)-[[alpha]]-DGalUA( 1-2). Rhamnogalacturonase A hydrolyses the glycoside bonds [[alpha]]-DGalUA(1-2)-[[alpha]]-L-Rha. The minimum size of the substrate has been found to be a 12'mer, resulting in a 5'mer and a 7'mer after enzymatic hydrolysis. Recombinant enzyme has been obtained from an overexpression system set up in Aspergillus oryzae. The enzyme crystallizes in space group I222 with one molecule in the asymmetric unit. The three dimensional fold of the enzyme consists almost entirely of parallel [[beta]]-strands wound into a right-handed [[beta]]-helix. There are twelve turns in the [[beta]]-helix, each comprising from one to three [[beta]]-strands, leaving one side of the molecule with all the loop regions. A large groove is found at this side, which could be the possible substrate binding site. The molecule is highly glycosylated, with two N-glycosylation sites and 18 O-glycosylation sites. All O-glycosylation sites are located in the C-terminal tail of the molecule (367 - 422), which is a long random coil element, that surrounds the molecule. The O-glycosylation seems to protect the long C-terminal tail from proteolytic degradation and its function is probably to keep the otherwise hydrophobic molecule in solution. Two other plant cell wall degrading enzymes Pel C and Pel E are known and though they share the same three dimensional fold as rhamnogalacturonase A, they are slightly smaller with about eight turns in the right-handed [[beta]]-helix.