CRYSTALLIZATION AND PRELIMINARY X-RAY STRUCTURE ANALYSIS OF D-DOPACHROME TAUTOMERASE. Hiroshi Sugimoto, Atsushi Nakagawa, Isao Tanaka, Jun Nishihira^*, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan, ^*Central Research Institutes, School of Medicine, Hokkaido University, Sapporo 060, Japan.

D-dopachrome tautomerase converts D-dopachrome into 5,6-dihydroxyindole (DHI). It was first observed in experiments when D-isomer of dopachrome was used as a control substrate for the study of isomerization of naturally occurring L-isomer in cultured melanoma cells. It is composed of 114 amino acids (Mr=12kDa). D-dopachrome tautomerase has no homology in amino acid sequence to L-dopachrome tautomerase. However it exhibits 27% sequence homology to macrophage migration inhibitory factor (MIF), a cytokine involved in inflammation and immune system. The three dimensional structure of MIF has been solved by ourselves. It has a similar trimeric b-prism structure to 5-carboxymethyl-2- hydroxymuconate isomerase (CHMI) from Escherichia coli. These findings prompted us to start the three dimensional structure analysis of D-dopachrome tautomerase.

D-dopachrome tautomerase has been crystallized at 18C by hanging drop vapor diffusion method. The reservoir solution contained 10% (w/v) PEGMME (polyethyleneglycol monomethylether) 2000, 0.1M Tris-HCl buffer (pH8.8) and 0.1M nickel chloride. Equal volumes of the reservoir solution and 20mg/ml protein solution in deionized water were mixed to give 8ml drops. Rod shaped crystals grew up to 0.3 x 0.3 x 0.8mm within a week. They diffract X-ray up to 3.0Å resolution. A preparation of selenomethionyl crystals for multiwavelength anomalous diffraction (MAD) method is under way.