Ternary Complex Formation and Induced Asymmetry in Orotate Phosphoribosyltransferase^,

Abstract

Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.

The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

“…6A). The observed difference in conformation is similar to the induced asymmetry observed in the S. cerevisiae OPRT structures (17). In addition to the conformational dissimilarity, a comparison of the LdOPRT active sites from the two chains in the LdUMPS structure revealed that there were differences in electron density.…”

“…The residues from the adjacent chain that participate are on a flexible loop region that is believed to close about the active site to occlude water and prevent unproductive reactions with the oxocarbenium ion intermediate (45,47,48). In addition to this loop movement, recent structures of Saccharomyces cerevisiae OPRT reveal an additional conformational change that occurs upon substrate binding (17). A rigid body rotation of a "hood" region was also observed in the complex of S. cerevisiae OPRT with OMP or orotate and PRPP (Fig.…”

“…OPRT is a transferase important for cancer chemotherapy as an activator of the prodrug 5-fluorouracil (16). There are also several available structures of OPRT (17)(18)(19) including a recent structure of the human OPRT domain of UMP synthase (PDB code 2WNS).…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…The structure of S. cerevisiae orotate phosphoribosyltransferase resembles that of S. enterica (230). As mentioned, the dimeric structure is asymmetric.…”

A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT

“…Like other phosphoribosyltransferases (20,27), arginine and lysine residues form ion pairs to pyrophosphate at the catalytic site. One of the Mg 2ϩ ions reduces the negative charge and links pyrophosphate to the active site and to the ribosyl group by interactions with (O␦1)D313, (F)BeF 3 Ϫ , O2R, O3R, OP␣(O5), and OP␤(O2).…”