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

摘要: The C-C bond cleavage catalyzed by metal-dependent iso-orotate decarboxylase (IDCase) from the thymidine salvage pathway is of interest for elucidation a (hypothetical) DNA demethylation pathway. IDCase appears also as promising candidate synthetic regioselective carboxylation N-heteroaromatics. Herein, we report joint experimental-theoretical study to gain insights into metal identity, reaction mechanism, and substrate specificity IDCase. In contrast previous assumptions, enzyme demonstrated ICPMS/MS measurements contain catalytically relevant Mn2+ rather than Zn2+. Quantum chemical calculations revealed that decarboxylation natural (5-carboxyuracil) proceeds via (reverse) electrophilic aromatic substitution with formation CO2. occurrence previously proposed tetrahedral carboxylate intermediates concomitant HCO3- could be ruled out on basis prohibitively high energy barriers. related o-benzoic acid decarboxylases, such γ-resorcylate 5-carboxyvanillate decarboxylase, which exhibit relaxed tolerance phenolic acids, shows fidelity. Structural comparisons suggest this caused unique hydrogen bonding heterocyclic surrounding residues. Analysis calculated energies reverse uracil impeded strongly disfavored uphill reaction.