5 research outputs found
Reaction Mechanism and Substrate Specificity of Iso-orotate Decarboxylase: A Combined Theoretical and Experimental Study
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 Îł-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
Adsorbent-Based Downstream-Processing of the Decarboxylase-Based Synthesis of 2,6-Dihydroxy-4-methylbenzoic Acid
In
this case study the regioselective enzymatic carboxylation of
3,5-dihydroxytoluene (orcinol) using the nonoxidative 2,3-dihydroxybenzoic
acid decarboxylase from <i>Aspergillus oryzae</i> (2,3-DHBD_Ao),
followed by an adsorbent-based downstream approach, has been investigated.
The product 2,6-dihydroxy-4-methylbenzoic acid (DHMBA) was herein
purified by an adsorptionâdesorption cycle and subsequently
obtained with purities >99% without a full elimination of the excess
bicarbonate from its reaction solution. Ten adsorbent resins were
studied in respect of their ability to recover the product from the
reaction solution, whereas the strong anion exchange resin Dowex 1x2
in its chloride form showed affinities >99%, even at bicarbonate
concentrations
of >3 mol·L<sup>â1</sup>. Desorption from loaded resin
was carried out by a 2 mol·L<sup>â1</sup> HCl/acetone
mixture, followed by product crystallization during acetone evaporation.
This presented concept does not require a final column preparation
step and improves the overall atom efficiency of the biocatalytic
reaction system
Pressurized CO<sub>2</sub><sup> </sup>as Carboxylating Agent for the Biocatalytic ortho-Carboxylation of Resorcinol
Utilization of gaseous carbon dioxide as a C1-building block in the biocatalytic ortho-carboxylation of a phenol.</p
Metal Ion Promiscuity and Structure of 2,3âDihydroxybenzoic Acid Decarboxylase of Aspergillus oryzae
Broad substrate tolerance and excellent regioselectivity, as well as independence from sensitive cofactors have established benzoic acid decarboxylases from microbial sources as efficient biocatalysts. Robustness under process conditions makes them particularly attractive for preparativeâscale applications. The divalent metalâdependent enzymes are capable of catalyzing the reversible nonâoxidative (de)carboxylation of a variety of electronârich (hetero)aromatic substrates analogously to the chemical KolbeâSchmitt reaction. Elemental mass spectrometry supported by crystal structure elucidation and quantum chemical calculations verified the presence of a catalytically relevant Mg complexed in the active site of 2,3âdihydroxybenoic acid decarboxylase from Aspergillus oryzae (2,3âDHBD_Ao). This unique example with respect to the nature of the metal is in contrast to mechanistically related decarboxylases, which generally have Zn or Mn as the catalytically active metal