ODCase inhibitors

A large number of additional structures have been reported since these original six structures were described in 2000 (Table 1) [18, 19, 20, 21]. These more recent structures fall into three groups (a) structures of ligand-free ODCase, (b) structures of wild-type ODCase bound to inhibitors, and (c) structures of ODCase mutants. 

Pai and coworkers also reported structural studies of several M. thermoau-totrophicum ODCase mutants (see Table 1) [19, 21]. These structural studies focused on how binding is affected when residues in the phosphate binding region, the recognition site for the 2- through 4-positions of the pyrimidine ring of bound inhibitors, and the Asp-Lys-Asp-Lys tetrad (see Fig. 4 and Table 2) are altered. 

In addition, although the close proximity of two aspartic acid residues to the likely position of the substrate carboxylate group observed in the reported crystal structures of ODCase (see Fig. 4 and Sect. 3) originally led to the hypothesis that electrostatic repulsion could lead to ground-state destabilization [16, 17], we feel that the various options available to enzymes to avoid such unfavorable interactions (such as carboxylate protonation), and the observed favorable interaction of the binding site with the negatively charged inhibitor BMP (see Fig. 4) render this mechanism unlikely [22]. 

Kollman and coworkers apphed a variety of computational methods to this mechanistic problem—including quantum mechanics on small model systems, molecular dynamics simulations with the AMBER force field on the whole ODCase-substrate system, and MM-PBSA free energy calculations on ODCase with bound OMP [38]. Based on their results, they proposed a decarboxylation mechanism for ODCase that involves C5 protonation. Their calculations at the MP2/6-31+G //HF/6-31+G level showed that C5 has a greater intrinsic proton affinity than C6, 02, and even 04. This, coupled with the fact that Lys72 (M. thermoautotrophicum numbering see Table 2) is near C5 and C6 in the inhibitor-bound crystal structures, prompted the authors to embrace a C5 protonation mechanism. However, the authors themselves acknowledged the uncertainties of their calculations because of approximations employed in representing the enzyme active site. 

Further efforts involving structural and kinetic experiments and making use of various mutants of ODCase have led to an improved understanding of the active site, even though it is still not possible to describe the exact chemical steps of the catalytic mechanism with complete certainty. In this review, the presently known crystal structures of ODCase, native as well as mutant forms, in their ligand-free form and in complex with various inhibitors will be discussed. The description will focus on the M. thermoautotrophicum ODCase because most of the structural work involves this enzyme also, it is the one the authors are most familiar with (and prejudiced towards). 

Fig. 1a,b Cartoon representation of the dimer of M. thermoautotrophicum ODCase. One monomer is in red and green, the other one in blue and yellow. Helices are displayed as ribbons and strands of /J-sheet as arrows. The 6-azaUMP inhibitor molecules (one per monomer) are shown in ball-and-stick representation, a View perpendicular to the twofold rotation axis relating the two monomers, b Side view, rotated by 90° compared to a, now looking down the rotation axis... 

Fig. 3 Structure-guided multiple sequence alignment of the four ODCases of known molecular structure. The fully conserved catalytic residues are highlighted in black. Other residues involved in inhibitor binding (Asp20, Serl27, Glnl85, Thr79 ) are highlighted in...

Fig. 4 Stereo representation of the interactions between active site residues in native M. thermoautotrophicum ODCase and the inhibitor 6-azaUMP. Several tightly bound water molecules are also shown. Blue hehces and yellow p-sheets belong to one monomer, red helices belong to the second monomer...

Fig. 6a,b Electron density representing the inhibitor of 6-azaUMP in the active sites of base-recognition mutants, a shows the dual conformations adopted by the pyrimidine ring in S127A ODCase. b In the Q185A mutant, a chain of water molecules replaces the glutamine side chain... 

Obviously, the inhibitor binds only weakly to the mutant enzyme and will diffuse out of the crystal lattice over time. This behavior can be explained when the interactions between this ligand and the mutant enzyme are analyzed in detail. Although the inhibitor occupies the exact same position as its counterpart in the native ODCase, there are only very few contacts left between nucleotide and protein matrix to hold the phosphate in place (Fig. 10). 

Fig. 2 The structures and binding affinities of eight competitive inhibitors of yeast ODCase. R-5 -P represents a 5 -phosphoribosyl group...

Several years after Beak and Siegel s proposal, Westheimer and coworkers synthesized 6-hydroxyuridine 5 -phosphate (BMP), a potent inhibitor (K,= 8.8x10 M) of yeast ODCase [15]. The structure of this competitive inhibi-... 

Fig. 5 Comparison of the binding affinities of yeast ODCase for OMP in the transition state for decarboxylation, orotate in the transition state for decarboxylation, and ribose 5 -phosphate acting as a competitive inhibitor. The loss of transition state binding energy obtained by cutting substrate OMP at the glycosidic bond totals more than 11 kcal/mol...

Despite various mechanistic studies over several decades and the recent crystal structures of ODCases from four microbial species in the presence of various inhibitors, the mechanism by which ODCase catalyzes decarboxylation of OMP remains unclear. At least five mechanisms for the generation of a more reactive intermediate have been proposed on the basis of different experimental and theoretical approaches (Fig. 1). In this review, the enzy-mological and model chemistry experiments designed to illuminate the nature of the more reactive ODCase intermediate will be surveyed. 

Suspecting that the preference of ODCase for anionic inhibitors might indicate the presence of a cationic active site amino acid residue, perhaps acting as a general acid in catalysis, Smiley and Jones sought to identify critical amino acid residues by site-directed mutagenesis, with particular attention to lysine residues [19]. By the late 1980s, quite a few ODCase sequences had... 

Fig. 3 Picture of the active site in ODCase showing only selected amino acids. The geometry of the active site is taken from the X-ray structure IDQX and the carboxylate group has been added to the inhibitor BMP. Selected residues have either been shown to be important for catalysis or play important roles in the computational models of this review. No hydrogens are included in the figure...

Wu and Pai provide a thorough examination of the molecular structure of ODCase, based on X-ray crystallographic studies of wild-type and mutant ODCases bound with various inhibitors. The implications of these structural studies are discussed, with an emphasis on the possibility that the conformational dynamics of the enzyme may be the key to catalysis. 

The anticancer nucleoside Gemcitabine as its monophosphate derivative (3) has been modified by conjugation of squalene at the N4-position. Using a combination of cryo-EM and SAXS it was shown that the squalenoyl derivative exists as a unilamellar liposome, and that the nanoassembly exhibited enhanced activity compared with Gemcitabine in a resistant cell line." Various C6-modified 2 -deoxy-2 -fluoro-dUMP derivatives have been prepared as potential inhibitors of orotidine 5 -monophosphate decarboxylase (ODCase) of these, the most potent analogue was the 6-iodo-derivative which was found to covalently inhibit ODCase. Aminoacyl-tRNAs... 

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