Abstract
The C-C bond cleavage catalyzed by metal-dependent iso-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 N-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 via a (reverse) electrophilic aromatic substitution with formation of CO2. 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 o-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.
Highlights
Iso-orotate decarboxylase (IDCase), an enzyme involved in the thymidine salvage pathway, catalyzes the non-oxidative decarboxylation of iso-orotate (5-carboxyuracil; 5caU; 1a) to uracil (U; 1b) (Smiley et al, 2005; Leal et al, 2007) (Scheme 1A)
Combined theoretical and experimental techniques have been employed in the present study to determine the metal identity, investigate the reaction mechanism and elucidate the substrate specificity of iso-orotate decarboxylase (IDCase)
ICPMS/MS measurements demonstrated the IDCase from C. militaris contains a catalytically relevant Mn2+ ion rather than the previously assumed Zn2+ ion
Summary
Iso-orotate decarboxylase (IDCase), an enzyme involved in the thymidine salvage pathway, catalyzes the non-oxidative decarboxylation of iso-orotate (5-carboxyuracil; 5caU; 1a) to uracil (U; 1b) (Smiley et al, 2005; Leal et al, 2007) (Scheme 1A). To investigate the reaction mechanism of IDCase, quantum chemical calculations were performed on the basis of the crystal structure of the Asp323Asn mutant from C. militaris in complex with the substrate (PDB 4LAM) (Xu et al, 2013).
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