Structures of the Human Orotidine-5′-Monophosphate Decarboxylase Support a Covalent Mechanism and Provide a Framework for Drug Design

The final two steps of de novo uridine 5-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

mentioning

confidence: 99%

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

French

Yates

Soysa

et al. 2011

“…Such a distortion effect was observed in crystal structures of various ODCase-ligand complexes, such as OMP, 6-aceto-UMP, and 6-cyano-UMP, with both MtODCase and human ODCase (11)(12)(13)(14). For 6-cyano-UMP, such a distortion receives strong support from Raman spectroscopy, which indicates bond bending of about 20° (15).…”

mentioning

confidence: 79%

Atomic Resolution Structure of the Orotidine 5′-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis

Fujihashi

Mito

Pai

et al. 2013

Background: Low binding affinity of product UMP is used to argue against substrate distortion contributing to orotidine-5Ј-monophosphate decarboxylase catalysis. Results: Atomic resolution structure and surface plasmon resonance analysis reveal strong repulsion between active site residue and UMP. Conclusion: Low UMP affinity does not disprove contribution of substrate distortion to catalysis. Significance: Substrate distortion can still be considered as a mechanistic feature of this most proficient enzyme.Orotidine 5-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of its substrate by 17 orders of magnitude. One argument brought forward against steric/electrostatic repulsion causing substrate distortion at the carboxylate substituent as part of the catalysis has been the weak binding affinity of the decarboxylated product (UMP). The crystal structure of the UMP complex of ODCase at atomic resolution (1.03 Å) shows steric competition between the product UMP and the side chain of a catalytic lysine residue. Surface plasmon resonance analysis indicates that UMP binds 5 orders of magnitude more tightly to a mutant in which the interfering side chain has been removed than to wild-type ODCase. These results explain the low affinity of UMP and counter a seemingly very strong argument against a contribution of substrate distortion to the catalytic reaction mechanism of ODCase.Orotidine 5Ј-monophosphate decarboxylase (ODCase) 3 is one of the most proficient enzymes known (1). It decarboxylates orotidine 5Ј-monophosphate (OMP) and produces uridine 5Ј-monophosphate (UMP) in the final step of the de novo pyrimidine biosynthesis pathway (Fig. 1A). It also accelerates the reaction by 17 orders of magnitude, as compared with the spontaneous reaction in water at neutral pH, without employing cofactors or metal ions (1-5).The reaction mechanism of this enzyme has been the subject of extensive investigations. More than 170 crystal structures have been determined, and numerous kinetic assays at various conditions have been performed. These experiments established that an electrostatic residue network composed of the charged side chains of two aspartate and two lysine residues, all completely conserved, plays a dominant role in catalysis (Fig. 1B) (2-5). As with all enzymes, the reaction acceleration provided by ODCase is explained in part by transition state stabilization (6). Lys-72 (the sequence numbers in this study correspond to those of the ODCase from Methanothermobacter thermautotrophicus (MtODCase)) is considered to be the key residue to stabilize the intermediate vinyl anion (6,7). However, there is still no general agreement on all the details of the reaction mechanism. In particular, the observation that ODCase also converts 6-cyano-UMP into 6-hydroxy-UMP at the same site where the decarboxylation reaction occurs (Fig. 1A) (8) complicates the scenario. The environment required to stabilize the vinyl anion does not seem suitable to support, at the same time, the intermediate of the side reaction b...

“…ODC activity in human cells is harbored in the C-terminal domain of the bifunctional UMPS enzyme that is also endowed with OPRT activity in its N-terminal domain (7). Human UMPS is an obligate dimer, and capping of one monomer by the other is strictly required to constitute a functional ODC catalytic center (22). The mechanism of decarboxylation involves tight binding and conformational restraint of the entire OMP molecule (phosphate, ribose, and base) in the active site to ensure substrate destabilization along the reaction coordinate by forcing the carboxylate group out of the plane of the pyrimidine ring.…”

Section: F-3durd Inhibits the Odc Activity Of Umps (Oprt/odc) Inmentioning

confidence: 99%

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

Balzarini

Gago

Kulik

et al. 2012

Background: 3-Deazauridine inhibits CTP synthetase as its 5Ј-triphosphate derivative and selectively depletes CTP pools. Results: 3-Deazauridine fluorination shifts its antimetabolic target from CTP-S to orotidylate decarboxylase, resulting in depletion of intracellular UTP and CTP. Conclusion: Such a target enzyme inhibition shift has a profound impact on the pyrimidine nucleotide pools. Significance: A hydrogen to fluorine replacement in an antimetabolite drug results in a target enzyme shift.