The primary goal of this research project is the elucidation of the three-dimensional structures and enzyme mechanisms of S- Adenosylmethionine (AdoMet) synthetase and S-adenosylhomocysteine (AdoHcy) hydrolase. This will be accomplished by determining and analyzing the crystal structures of native enzymes as well as the structures of complexes between these enzymes and substrates (or inhibitors). Because of the important role that AdoMet mediated transmethylations play in regulating cellular events, the enzymes involved in these transmethylations are attractive targets for the design of potential chemotherapeutic agents. Both AdoMet synthetase and AdoHcy hydrolase seems to be key enzymes to maintain the transmethylation reactions. Prior to the rational design of inhibitors for these enzymes, the structural data which are the target of the proposed research must be obtained. Adequate amounts of AdoMet synthetase from Escherichia coli and AdoHcy hydrolase from Alcaligenes faecalis have been provided by collaborators, Dr. G.D. Markham and Professor S. Shimizu, respectively. We can routinely grow relatively large hexagonal bipyramidal crystals (approximately 1.0 mm) of AdoMet synthetase. The crystals belong to the hexagonal system with space group P6222 or P6422 and cell dimensions a=128.8, c=140.3 angstrom. One subunit (Mr=42.000, 384 amino-residues) of the tetrameric enzyme is in a crystallographic asymmetric unit. The crystals diffract to about 3.0 angstrom resolution using a conventional X-ray generator at room temperature. 3.5 angstrom resolution data of native and 3.7 angstrom resolution data of a UO2Cl2 derivative including anomalous scattering data were collected by using an area detector X-ray diffractometer at the Biotechnology Resource of the University of Virginia. A difference Patterson map calculated by using 5 angstrom resolution data clearly shows the major and minor sites of UO2. A search for another good heavy atom derivative is in progress. AdoHcy hydrolase is a hexamer composed of six identical subunits, molecular weight 48,000 daltons. Crystals with tetragonal shapes and thin plates are obtained. The tetragonal shaped crystals could be suitable for the crystallographic studies, but are slightly too small. Efforts to grow the large crystals are in progress.
