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

Abstract

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...

Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-Dependent and Cofactor-Independent Examples.

“…145 On the basis of experiments with a 6-phosphonic acid derivative of uridine-5'-phosphate, as an analogue of the substrate that cannot be decarboxylated, the authors claimed to also have support for the substrate destabilization idea, 146 as did a recent X-ray study at 1.0 Å resolution with product bound. 147 …”

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

“…In addition, our WT-UMP complex structure, recently determined at atomic resolution (1.03 Å), indicates that the pyrimidine ring of UMP itself is slightly distorted, too. 45 These structures imply that ODCase can utilize substrate distortion to achieve the enormous acceleration of the reaction it catalyzes. Although various groups have undertaken computational simulations of ODCase catalysis, detailed analyses of the distortion effects have not been performed thus far.…”

“…These findings clearly invalidate the original argument. 45 The disruption of the resonance between the rotated carboxylate group and the pyrimidine ring might ease the distortion. The C7 atom moves out of the pyrimidine ring plane, the planarity of which is also disrupted.…”

An Investigation of the YidC-Mediated Membrane Insertion of Pf3 Coat Protein Using Molecular Dynamics Simulations

“…The crystal structure of YidC (PDB:3WO7 40 ) was downloaded from the Protein Data Bank. Initially, the system was prepared using the Molecular Operating Environment (MOE) software 41 by removing the water molecules from the crystal structure and assigning the appropriate protonation states for the residues using the protonate3D facility.…”