STRUCTURE AND CRYSTAL PACKING STUDIES OF 4-OXALOCROTONATE TAUTOMERASE. Alexander B. Taylor*, Christian P. Whitman, and Marvin L. Hackert* *Department of Chemistry & Biochemistry, The University of Texas at Austin, Austin, Texas 78712, Medicinal Chemistry Division, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712
4-Oxalocrotonate tautomerase (4-OT) is a highly efficient enzyme with an unusual mode of catalysis and an unexpected crystal packing scheme. 4-OT takes part in the meta-fission pathway encoded by the Pseudomonas putida mt-2 TOL plasmid pWW0 for catabolism of toluene, m- and p-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene to Krebs cycle intermediates.1 4-OT catalyzes the isomerization of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the intermediate, 2-hydroxymuconate. The structure has been solved to 2.5 resolution by the Molecular Replacement method with a recombinant 4-OT isozyme from Pseudomonas sp. Strain CF600 serving as a model.
4-OT is a hexamer composed of small monomers of 62 amino acids arranged with 32 symmetry.2 The monomer consists of a two-stranded parallel [[beta]]-sheet with a linking helix; dimerization leads to a four-stranded sheet with antiparallel helices on one side. Each dimer contains two active sites with an unusual catalytic amino-terminal proline.3 The active site has residues contributed from both subunits of the dimer.
4-OT crystallizes in space groups R3 (a=98.7, "=52.5, 6 dimers/a.u.) and P321 (a=88.0, c=124.6, 4.5 dimers/a.u.). In both crystal systems, the crystal packing is dictated by a shifted stack of three hexamers. Current work includes cocrystallization of native enzyme with mechanism-based inhibitors.
1Chen L. et al. (1992) JBC. 267, 17716-17721.
2Subramanya, H.S. et al. (1996) Biochemistry. 35, 792-802.
3Stivers, J.T. et al. (1996) Biochemistry. 35, 803-813.