X-RAY STRUCTURE OF INOSINE MONOPHOSPHATE DEHYDROGENASE FROM THE PROTOZOAN PARASITE TRITRICHOMONAS FOETUS. Frank G. Whitbyt^{+[[paragraph]]}, Hartmut Luecke^{[[daggerdbl]]}, John Somoza⁺, Hiro Tsaruta^{[[daggerdbl]]}, Jorge Huete-Perez⁺, Christopher P. Hill^{[[paragraph]]}, Robert J. Fletterick^+, Ching Chung Wang^{+, +}Department of Pharmaceutical Chemistry, University of California, San Francisco, California ^{[[daggerdbl]]}Stanford Synchrotron Radiation Laboratory, Stanford University, Palo Alto, California ^{[[paragraph]]}Department of Biochemistry, University of Utah Medical Center, Salt Lake City, Utah 84132

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the NAD-dependent oxidation of inosine monophosphate (IMP) to xanthine monophosphate (XMP). This is the rate-limiting step in purine biosynthesis. Inhibitors of this enzyme have been shown to have anti-tumor, immunosuppressive, and antiparasitic effects.

The enzyme is a tetramer of 230 kD total weight. Crystals of the recombinant enzyme were grown in 2.2 M ammonium sulfate and crystallized in the cubic space group P432. Data were collected at beamlines 1-5 and 7-1 at SSRL, and at beamline F-1 at CHESS. Initial low-resolution SIR and MIR electron density maps indicated a flat, tetrameric molecule, consistent with the expected crystal packing with one monomer per asymmetric unit. Solvent flattening was performed with the program PHASES. From the solvent flattened maps, about 280 alanine residues were fit in an alpha(8)-beta(8) barrel (TIM barrel). Phase combination with the MIR and refined partial model phases was performed. Heavy-atom difference maps indicate clearly 5 cysteine residues that reacted with either PCMBS or thimerosal, and 3 methionine residues that bound platinum chloride. One disulfide bond has been discovered and may be related to the observed increase in activity of this enzyme in the presence of reducing agent. In addition, the active site has been identified by the position of an active-site cysteine and the positions of electron density in difference maps calculated from data collected from ligand and inhibitor-soaked crystals. Presently the crystallographic R-factor is 21% to 2.0 angstroms resolution for data greater than 2 sigma, and R-free is 27%. The model will hopefully serve as a model for solving the structure of the human enzyme and in the design of inhibitors.