Reaction Mechanism and Substrate Specificity of -orotate Decarboxylase: A Combined Theoretical and Experimental Study.

The C-C bond cleavage catalyzed by metal-dependent -orotate decarboxylase (IDCase) from the thymidine salvage pathway is of interest for the elucidation of a (hypothetical) DNA demethylation pathway. IDCase appears also as a promising candidate for the synthetic regioselective carboxylation of -heteroaromatics. Herein, we report a joint experimental-theoretical study to gain insights into the metal identity, reaction mechanism, and substrate specificity of IDCase. In contrast to previous assumptions, the enzyme is demonstrated by ICPMS/MS measurements to contain a catalytically relevant Mn rather than Zn. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds a (reverse) electrophilic aromatic substitution with formation of CO. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of could be ruled out on the basis of prohibitively high energy barriers. In contrast to related -benzoic acid decarboxylases, such as γ-resorcylate decarboxylase and 5-carboxyvanillate decarboxylase, which exhibit a relaxed substrate tolerance for phenolic acids, IDCase shows high substrate fidelity. Structural and energy comparisons suggest that this is caused by a unique hydrogen bonding of the heterocyclic natural substrate (5-carboxyuracil) to the surrounding residues. Analysis of calculated energies also shows that the reverse carboxylation of uracil is impeded by a strongly disfavored uphill reaction.