THE CRYSTAL-STRUCTURE OF THE OROTATE PHOSPHORIBOSYLTRANSFERASE COMPLEXED WITH OROTATE AND ALPHA-D-5-PHOSPHORIBOSYL-1-PYROPHOSPHATE

The three-dimensional structure of Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base erotic acid has been solved and refined with X-ray diffraction data extending to 2.3 Angstrom resolution to a crystallographic R-factor of 18.7%. The complex was generated by carrying out catalysis in the crystal. Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] revealed that the enzyme backbone undergoes only small movements. The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5'-phosphate binding loop, and a long, solvent-exposed loop at the dimer interface. The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP . orotate complexes, with its anomeric carbon moving approximately 7 Angstrom across the binding cavity. In the PRPP . orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate wiht the substrate in the OMP complex.