Structural Basis for the Catalytic Mechanism of a Proficient Enzyme:  Orotidine 5‘-Monophosphate Decarboxylase^,

Abstract

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction rate by a factor of 10(17). This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups.

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

“…11 Lys44 may also serve, along with Lys73, to balance the charges of Asp71 and 76 in the region of the active site where the substrate carboxylate binds. 11 These roles could be satisfied by an arginine at position 44, which may explain why arginine was also found at this position among functional mutants. Lys73 also interacts with Asp71 and may serve to balance the charge of the Asp residues as described for Lys44.…”

“…The 24 residues chosen for randomization were picked based on their location relative to the barbituric acid monosphosphate (BMP) inhibitor bound in the E. coli ODCase active site crystal structure (PDB code: 1EIX). 11 The residues chosen are in direct contact with the inhibitor or are in the second shell around the residues in contact with the inhibitor (Fig. 3).…”

“…5). 11,16 Because of the importance of these positions in the catalytic mechanism, it was expected that only aspartate would be selected for function among random mutants at positions 71 and 76. Consistent with expectations, aspartate was found most often among functional mutants from the 71 and 76 libraries (Fig.…”

“…These values are similar to those obtained previously for the E. coli enzyme as well as the Mt and P. falciparum ODCases while K m is somewhat higher (sixfold) than observed for the yeast enzyme. 9,11,29,32 The D71C enzyme exhibited an 84-fold decrease in k cat and a 1071-fold decrease in catalytic efficiency compared with the wild-type enzyme (Table I). This indicates that mutants exhibiting as low as 0.1% of wild-type activity are recovered in the plasmid Figure 5.…”

“…8,9 The X-ray crystal structures of ODCase from several organisms including human, parasite, yeast, Bacillus subtilis and E. coli, among others, have been determined. 10,11 The mechanism by which ODCase achieves such a large rate enhancement over the noncatalyzed rate has been a subject of active investigation. 12 There are several reports that strongly support the existence of a substrate C6 carbanion intermediate during catalysis.…”