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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 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 HCO3- could be ruled out on the basis of prohibitively high energy barriers. In contrast to related o-benzoic acid decarboxylases, such as ?-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.

SUBMITTER: Sheng X 

PROVIDER: S-EPMC6305744 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Sheng Xiang X   Plasch Katharina K   Payer Stefan E SE   Ertl Claudia C   Hofer Gerhard G   Keller Walter W   Braeuer Simone S   Goessler Walter W   Glueck Silvia M SM   Himo Fahmi F   Faber Kurt K  

Frontiers in chemistry 20181219


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 t  ...[more]

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