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 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.

Non‐Oxidative Enzymatic (De)Carboxylation of (Hetero)Aromatics and Acrylic Acid Derivatives

“…Density functional theory (DFT) calculations employing large active site models based on crystal structures of ortho ‐benzoic acid decarboxylases ( o ‐BDCs) strongly support a general mechanistic proposal, which resembles a (reverse) electrophilic aromatic substitution by feasible energy barriers and bears a strong resemblance to the Kolbe–Schmitt reaction . In more detail, this general mechanism involves the metal ion (predominantly manganese or zinc) chelating the carboxylate and phenolate group of the arene substrate, thereby stabilizing their negative charge.…”

“…They share significant structural and mechanistic similarities, in particular the characteristic (β/α) 8 ‐barrel fold harbouring one catalytically relevant divalent metal ion in the active site . Whereas the overall sequence similarity between distinct subclasses is rather low (around 30%), several amino acid residues relevant for catalysis are conserved. Although members of the AHS commonly catalyze the hydrolysis of ester and amide bonds attached to either a carbon or phosphorus atom on a wide range of structurally diverse substrates, some members obviously evolved to catalyze the reversible decarboxylation of benzoic acid derivatives and nitrogen‐heterocyclic derivatives thereof.…”

“…Coordination arranges the nucleophilic phenolate for protonation at the carboxylate ipso ‐carbon by a nearby conserved catalytic acid (Asp), which is accompanied by dearomatization of the arene. The latter is restored upon loss of carbon dioxide, which dissociates together with the decarboxylated phenol from the metal center (Scheme ) …”

“…In contrast, the biocatalytic characterization of another member of this class of enzymes – iso ‐orotate decarboxylase (IDCase) – acting on a heterocyclic substrate analogue, revealed a very narrow substrate specificity and only acts in the energetically favoured decarboxylation direction …”

Metal Ion Promiscuity and Structure of 2,3‐Dihydroxybenzoic Acid Decarboxylase of Aspergillus oryzae

“…In order to facilitate their applicability, computational methods are increasingly applied for the prediction of substrate‐structure activities aiming to minimize time‐consuming and expensive trial‐and‐error wet‐lab experiments. Although the mechanism of o ‐BDs is basically well understood, [12,13] conflicting data exist concerning the nature of their catalytically essential divalent metal, for example, Zn 2+ , Mn 2+ or Mg 2+ , which aggravates computational studies leading to inaccuracies in substrate‐binding and energy pathways depending on the ionic radius and Lewis acidity [14] of the metal involved. In this study, we investigated the metal dependence of 2,3‐dihydroxybenzoate decarboxylase from Aspergillus oryzae (2,3‐DHBD_ Ao ) [15] by determination of its crystal structure, metal ion analysis by inductively coupled plasma tandem mass spectrometry (ICPMS/MS) and the catalytic energy profile by quantum chemical calculations.…”