Lys314 is a Nucleophile in Non‐Classical Reactions of Orotidine‐5′‐Monophosphate Decarboxylase

Abstract

Orotidine-5'-monophosphate decarboxylase (OMPD) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP) in an extremely proficient manner. The reaction does not require any cofactors and proceeds by an unknown mechanism. In addition to decarboxylation, OMPD is able to catalyze other reactions. We show that several C6-substituted UMP derivatives undergo hydrolysis or substitution reactions that depend on a lysine residue (Lys314) in the OMPD active site. 6-Cyano-UMP is converted to UMP, and UMP derivatives with good leaving groups inhibit OMPD by a suicide mechanism in which Lys314 covalently binds to the substrate. These non-classical reactivities of human OMPD were characterized by cocrystallization and freeze-trapping experiments with wild-type OMPD and two active-site mutants by using substrate and inhibitor nucleotides. The structures show that the C6-substituents are not coplanar with the pyrimidine ring. The extent of this substrate distortion is a function of the substituent geometry. Structure-based mechanisms for the reaction of 6-substituted UMP derivatives are extracted in accordance with results from mutagenesis, mass spectrometry, and OMPD enzyme activity. The Lys314-based mechanisms explain the chemodiversity of OMPD, and offer a strategy to design mechanism-based inhibitors that could be used for antineoplastic purposes for example.

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

“…Although the distortion is not very obvious in the present ester complexes due to overlapping electron densities, the bonds linking the C6 atoms and the respective substituents of 6-methyl-UMP, 6-cyano-UMP and 6-acetyl-UMP are clearly distorted in their ODCase complexes (Fig 2A and refs. 15,20 ). 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.…”

“…17,18 Similarly distorted ligands were found in the structures of several compounds bound to human-ODCase ( Hs ODCase) 19,20 and it has been suggested that the size of the C6-substituents of the pyrimidine ring is the dominant factor in determining the extent of the observed deviation from low energy conformations. 20 As the stable existence of such distorted structures strongly argues for the involvement of substrate distortion in ODCase catalysis we engaged in further analysis and investigated the crystal structures of four more complexes, i.e. the structures of 6-methyl-UMP, 6-amino-UMP, OMP-methyl-ester, and OMP-ethyl-ester (Fig.…”

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

“…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).…”

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

“…The loss of entropy also affects nucleotide analogues that inhibit ODC, which likely explains why inhibition constants are all in the high micromolar range (except for those inhibitors that bind covalently to the enzyme, e.g. 6-iodo-UMP) (26).…”

“…It also has a hydrogen bond donor (amino group) at C4 analogous to the phenolic hydroxyl group at C4 of 3F-3DUrd. It is possible that 3F-3DUrd binds to human ODC by occupying its active site in a different manner than other potent inhibitors such as 5Ј-phosphoribofuranosyl barbituric acid and 6AUrd that are anchored through an extended hydrogen-bonded network involving all of the phosphate oxygens, the ribose hydroxyl groups, and the nucleobase (26).…”