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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Chem. | doi: 10.3389/fchem.2018.00608

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

 Xiang Sheng1, Katharina Plasch2, Stefan E. Payer2,  Claudia Ertl2,  Gerhard Hofer2,  Walter Keller2,  Simone Braeuer2, Walter Goessler2,  Silvia M. Glueck2,  Fahmi Himo1* and Kurt Faber2*
  • 1Stockholm University, Sweden
  • 2University of Graz, Austria

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 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.

Keywords: Computational Chemistry, Biocatalysis, iso-orotate decarboxylase, reaction mechanism, Substrate Specificity, Metal identity

Received: 26 Sep 2018; Accepted: 27 Nov 2018.

Edited by:

Rajeev Prabhakar, University of Miami, United States

Reviewed by:

Stacey Wetmore, University of Lethbridge, Canada
Feliu Maseras, Institut Català d'Investigació Química, Spain  

Copyright: © 2018 Sheng, Plasch, Payer, Ertl, Hofer, Keller, Braeuer, Goessler, Glueck, Himo and Faber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. Fahmi Himo, Stockholm University, Stockholm, Sweden, fahmi.himo@su.se
Prof. Kurt Faber, University of Graz, Graz, 8010, Styria, Austria, Kurt.Faber@Uni-Graz.at