ODCase crystal structure

The ODCase crystal structures reveal a novel array of charged residues located in a region of the active site where the substrate s reactive carboxylate is... 

The mechanism of the enzymatic decarboxylation of orotidine 5 -mono-phosphate (OMP) to uridine 5 -monophosphate (UMP) (see Fig. 1) is an intriguing problem for which many solutions have been offered. Even before 1995 when Wolfenden and Radzicka declared OMP decarboxylase (ODCase) to be the most proficient enzyme [1], several different mechanisms had been proposed. Since that time, other mechanisms have been advocated. Curiously, the appearance of crystal structures for various wild-type and mutant ODCases has led not to a definitive picture of catalysis, but to even more conjecture and controversy concerning the mechanism. 

To date, more than twenty X-ray crystal structures of ODCase have been reported, all by the groups of Ealick, Pai, Larsen, and Short and Wolfenden (see Table 1) [14-21]. The first six of these were reviewed previously by Houk, Lee, and coworkers [22] and Begley, Appleby, and Ealick [23]. As noted by these reviewers, the active-site structure of ODCase is extremely similar in all of the reported structures, boasting an Asp-Lys-Asp-Lys tetrad in the vicinity of the 6-position of the pyrimidine... 

Table 1 X-ray crystal structures of ODCase, listed in the order reported... 

After the initial crystal structures of ODCase were reported, a modified 04 protonation mechanism was proposed [22]. In the original Lee-Houk proposal, the active-site residue Lys93 (yeast numbering, analogous to Lys72 in E, coli Fig. 4 and Table 2), which was experimentally shown to be catalyt-ically important, was assumed to be the species that protonated 04 [27, 32]. However, in the reported crystal structures this lysine does not reside near 04. It was therefore proposed that proton transfer from Lys93 to 04 might be mediated by an active site water molecule (see Fig. 7). The viability of this scenario has not yet been established, however. 

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. 

Despite intense experimental and theoretical efforts, the mystery surrounding the proficiency of ODCase has not yet been solved. This summary of the experimental facts and their imphcations, analysis of the structural data available from various crystal structures, description of the various computational studies performed on this problem, and discussion of the important mechanisms that have been proposed, shows the exciting challenges that remain in understanding the action of one of nature s most proficient enzymes. 

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). 

Lee and Houk calculated gas phase proton affinities of orotate and depro-tonated uracil, which suggest that 0-4 rather than 0-2 is the favorable site of protonation for substrate OMP [24]. On the basis of these findings, Lee and Houk proposed a carbene-based mechanism that involves protonation at 0-4 by either an active-site acidic residue or a site-bound water molecule (Fig. 3c) [24, 25]. In this mechanism, the formation of a neutral carbene at C-6 is stabilized by an active site environment that displays a low dielectric constant. The recent determination of the crystal structures of ODCase (see below) questions the plausibility of this mechanism. These structures reveal a highly charged active site, one that might be poorly suited for stabilization of an uncharged carbene. The structures also demonstrate the lack of an acidic residue near the 0-4 atoms of bound ligands. 

Based on the crystal structure of the Bacillus subtilis enzyme in complex with product UMP, Appleby and associates have proposed a novel, electrophilic substitution mechanism for the ODCase reaction (Fig. 3d) [26]. Instead of requiring the formation of a discrete carbanion in the transition state for decarboxylation, this mechanism postulates that Lys-93 protonates... 

Recently, the crystal structures of ODCase from yeast and from three different bacterial sources have been reported [26, 31, 32, 33]. Although a detailed comparison of the available crystal structures is beyond the scope of this review, the structure of the yeast enzyme both free and in complex with the postulated transition state analog, BMP, will be briefly discussed. For a detailed discussion of the currently available crystal structures, readers are referred to reference [34] and the article by Wu and colleagues in this issue. 

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. 

In an effort to shine more light onto this puzzling question, we co-crystal-lized native ODCase with XMP and CMP, respectively. The structural analyses of the resulting complex crystals revealed a second mode of binding. Whereas the phosphates occupy the same place as in all other complexes, the ribose groups are moved somewhat from the positions they adopt in the UMP, 6-aza-... 

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