The crystal structure and mechanism of orotidine 5'-monophosphate decarboxylase

Abstract

The crystal structure of Bacillus subtilis orotidine 5'-monophosphate (OMP) decarboxylase with bound uridine 5'-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R-factor of 19.3% at 2.4 A resolution. OMP decarboxylase is a dimer of two identical subunits. Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface. For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer. The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism. The uridine 5'-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried. In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60. This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62. We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction.

Scheme 1

Figure 1

Experimental electron density at 2.5 Å resolution (unaveraged) for UMP of monomer C using solvent flattened multiple wavelength anomalous diffraction phases. The contour level is 1σ.

Figure 2

Ribbon drawing of the OMP decarboxylase monomer. Helices and strands are labeled, and UMP is shown as a ball-and-stick model. The drawing was prepared with molscript (26).

Figure 3

Topology diagram showing the OMP decarboxylase fold. The first and last residue numbers are given for each α-helix and β-strand.

Figure 4

Stereodiagram showing the OMP decarboxylase dimer. Different colors are used for each subunit, and UMP is shown as a ball-and-stick model. The drawing was prepared with molscript (26).

Figure 5

Active site of OMP decarboxylase. The stereoview shows UMP and the key amino acid residues. The red spheres represent water molecules. The drawing was prepared with ribbons (29).

Figure 6

Schematic representation of the OMP decarboxylase active site. Only charged and hydrophilic residues are included. The carboxylate of OMP is based on modeling studies. Hydrogen bonds are shown as dashed lines.

Figure 7

Proposed reaction mechanism for OMP decarboxylase. (A) Schematic representation of the reaction with the proposed transition state. (B) Stereoview of a model of the enzyme substrate complex. The OMP was generated by modeling a carboxylate group at C6 coplanar to the pyrimidine ring of UMP. The coordinates of UMP, Asp60, and Lys 62 were used without modification from the crystal structure.