AUTHOR=Sheng Xiang , Plasch Katharina , Payer Stefan E. , Ertl Claudia , Hofer Gerhard , Keller Walter , Braeuer Simone , Goessler Walter , Glueck Silvia M. , Himo Fahmi , Faber Kurt 

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

JOURNAL=Frontiers in Chemistry

VOLUME=6

YEAR=2018

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2018.00608

DOI=10.3389/fchem.2018.00608

ISSN=2296-2646

ABSTRACT=<p>The C-C bond cleavage catalyzed by metal-dependent <italic>iso</italic>-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 <italic>N</italic>-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 Mn<sup>2+</sup> rather than Zn<sup>2+</sup>. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds <italic>via</italic> a (reverse) electrophilic aromatic substitution with formation of CO<sub>2</sub>. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of <inline-formula><mml:math id="M1" 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></inline-formula> could be ruled out on the basis of prohibitively high energy barriers. In contrast to related <italic>o</italic>-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.</p>