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. 

Simultaneous mutation of Lys72 and Asp70 both to Ala resulted in the first reported ODCase-OMP complex [19]. In this complex, the carboxylate of OMP, along with a water molecule, occupies space normally occupied by 

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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. 

Fig. 44 Pathways for uridylate biosynthesis. Mutants lacking enzymes PRTase or ODCase can complete a route to UMP provided by an antibody orotate decarboxylase in conjunction with the naturally occurring uracil PRTase. Decarboxylation of orotic acid [135] is thought to proceed through the transition state [136], for which the hapten [137] was developed (Smiley and Benkovic, 1994).

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. 

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. 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... 

Whether it is the central position proposed for Asp70 in the electrostatic stress mechanism [12] or the role of Lys72 as proton-donor to the carbanion intermediate [24], it is obvious that these two residues are the most important ones in catalysis. Nevertheless, all Asp70 as well as Lys72 single-site mutants of M. thermoautotrophicum ODCase that were generated still retained some enzymatic activity (see below), a clear indication that it is not a sole residue carrying all of the catalytic load [21]. 

Fig. 9 Crystals of the 6-azaUMP complex of the AR203A mutant of M. thermoautotroph-icum ODCase. Older crystals have bent and crystals of apo-enzyme (small diamonds) grow on their tips...

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). 

The relative contribution of each interaction was calculated from the ratio of the ligand dissociation constant determined with the mutant enzyme to that determined with wild-type yeast ODCase... 

Table 3 Summary of the kinetic properties of wild-type and mutant yeast ODCases ...

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. 

Miller provides a synthesis of results from recent structural studies, computations, and biochemical experiments on mutant ODCases and truncated substrates. His detailed analysis of specific ODCase-substrate interactions is aimed at quantifying their importance and proposing roles for each in catalysis. 

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