MODELING OF THE SPATIAL STRUCTURE OF EUKARYOTIC ORNITHINE DECARBOXYLASES. Nick V. Grishin, Margaret A. Phillips, Elizabeth J. Goldsmith, University of Texas Southwestern Medical Center, Dallas, TX, 75235

Based on sequence analysis the pyridoxal phosphate (PLP) dependent enzyme eucaryotic ornithine decarboxylase (ODC) is predicted to have a [[beta]]/[[alpha]] barrel fold. We found that the sequences of reported PLP dependent enzymes (313 in total) fall into seven fold types, three of which were previously described crystallographically . ODC and alanine racemases were shown to be related and display 15% identity. These enzymes have no detectable sequence identity with any protein of known spatial structure, but match secondary structure and hydrophobicity profiles of a [[beta]]/[[alpha]]-barrel template. Through the analysis of known barrel structures we developed a topographic model of ODC active site. Out of 11 active site residues in glycolate oxidase, an FMN-dependent enzyme with known structure, 9 are invariant in the ODC model. Our model predicts the phosphate group of the PLP is located between the C-termini of the seventh and eighth strands, bound to a Gly-rich region in the loop after strand 7 and the N-terminus of a small helix with Arg277 after strand 8. The model suggests that Glu274 or Asp233 may interact with the pyridoxal nitrogen. Characterization of the Glu274 to Ala mutant Trypanosoma brucei ODC demonstrated that Glu274 functions to stabilize the positive charge on the pyridoxal nitrogen, and Arg277 to Ala mutant displayed lower affinity to PLP, supporting the model. Crystals of Tryponosoma brucei ODC were obtained (P2₁, a=67, b=152, c=85, [[beta]]=103; R_m 7.5%, 99% complete to 3Å resolution). The work on phase determination is in progress.