THE RELATION BETWEEN DIFFERENT POLYMORPHIC MODIFICATIONS OF RHAMNOGALACTURONAN ACETYLESTERASE. Anne B. Gjerlov and Sine Larsen, Centre for Crystallographic Studies, University of Copenhagen, Denmark

Rharnnogalacturonan acetylesterase (RGAE) is a novel enzyme involved in the the ezymatic degradation of the polysaccharide rhamnogalacturonan, one of the major components of pectin. RGAE has been isolated from Aspergillus aculeatus and overexpressed in Aspergillus oryzueⁱ. The structure determination of the enzyme showed that it has an open twisted [[alpha]]/[[beta]] fold.

The protein crystallizes readily, despite substantial glycosylation, and has been crystallized under several different conditions, yielding three different crystal forms. Using the hanging drop method and the conditions 19% PEG 4000, 18% isopropanol, 40 mg/ml RGAE and 0.1M citrate buffer, a tetragonal form is obtained around pH 4.1, an orthorhombic form around pH 4.7 and a trigonal form at pH 5.1. The orthorhombic crystals can also be obtained with the conditions 1.4M (NH₄)₂SO₄ or 1.4M Li₂SO₄, 0.1M Na acetate, 40 mg/ml RGAE and pH 5.0.

The structure of the orthorhombic form was solved by Multiple Isomorphous Replacement, using two heavy atom derivatives. The spacegroup is P2₁2₁2₁ with cell parameters a=52.14Å, b=56.87Å, c=71.89Å. There is one molecule in the asymmetric unit and the water content is 35%. Data collection was performed with a rotating anode as X-ray source operating at room temperature. The structure was refined using data to a resolution of l.55Å. The results from investigations of the two other modifications by the molecular replacement method will also be presented and related to the difference in crystallization conditions.

ⁱKauppinen, S., Christgau, S., Kofod, L.V., Halkier, T., Dörreich, K. and Dalbge, H. (1995) J.Bio.Chem., 270, 27172-27178.