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

Abstract

The C-C bond cleavage catalyzed by metal-dependent <i>iso</i>-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 <i>N</i>-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 Mn²⁺ rather than Zn²⁺. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds <i>via</i> a (reverse) electrophilic aromatic substitution with formation of CO₂. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:msubsup><mml:mrow><mml:mtext>HCO</mml:mtext></mml:mrow> <mml:mrow><mml:mn>3</mml:mn></mml:mrow> <mml:mrow><mml:mo>-</mml:mo></mml:mrow> </mml:msubsup> </mml:math> could be ruled out on the basis of prohibitively high energy barriers. In contrast to related <i>o</i>-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.