10 research outputs found
Substrate-based protein engineering of a flavoprotein oxidase for improved alcohol over-oxidation
The oxidation of alcohols to the corresponding carbonyl compounds represents a convenient strategy for the selective introduction of carbon-acceptor units into carbohydrate-based starting materials from renewable resources. A simple system to accomplish this transformation is by using flavin-containing alcohol oxidases. However, with prim-alcohols, the oxidation does not necessarily stop at the aldehyde stage, but may furnish the carboxylic acid via \u27over\u27-oxidation of the aldehyde hydrate.[1] In order to develop an alcohol oxidase for the efficient transformation of alcohols into carboxylic acids, we chose the recently discovered (5-hydroxymethyl)furfural oxidase (HMFO), which converts not only the eponymous (5-hydroxymethyl)furfural, but also a range of aromatic and allylic alcohols (Figure 1).[2]In order to improve the performance of HMFO for over-oxidation, we anticipated an improved stabilisation of the aldehyde hydrate in the active site to be a crucial factor. After inspection of the HMFO crystal structure, two positions were identified, where hydrogen bond donating and accepting amino acids were introduced, in order to stabilize the gem-diol moiety.[3] Indeed, one of the new HMFO variants exhibited a significantly increased activity for the formation of carboxylic acids from benzylic alcohols.
Please click Additional Files below to see the full abstract
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
Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids
The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to âover-oxidationâ of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and MichaelisâMenten kinetics were performed to corroborate these findings
A Rational Active-Site Redesign Converts a Decarboxylase into a Cî»C Hydratase: âTethered Acetateâ Supports Enantioselective Hydration of 4âHydroxystyrenes
The
promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes
catalyzed by ferulic acid decarboxylase from <i>Enterobacter</i> sp. (FDC_<i>Es</i>) depends on bicarbonate bound in the
active site, which serves as a proton relay activating a water molecule
for nucleophilic attack on a quinone methide electrophile. This âcofactorâ
is crucial for achieving improved conversions and high stereoselectivities
for (<i>S</i>)-configured benzylic alcohol products. Similar
effects were observed with simple aliphatic carboxylic acids as additives.
A rational redesign of the active site by replacing the bicarbonate
or acetate âcofactorâ with a newly introduced side-chain
carboxylate from an adjacent amino acid yielded mutants that efficiently
acted as Cî»C hydratases. A single-point mutation of valine
46 to glutamate or aspartate improved the hydration activity by 40%
and boosted the stereoselectivity 39-fold in the absence of bicarbonate
or acetate
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
Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives
The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed <i>o</i>-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the ÎČ-carbon atom of styrenes forming (<i>E</i>)-cinnamic acids
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
Terminal Alkenes from Acrylic Acid Derivatives via Non-Oxidative Enzymatic Decarboxylation by Ferulic Acid Decarboxylases
Regioselective para-Carboxylation of Catechols by a Prenylated Flavin Dependent Decarboxylase
The utilization of CO as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN4 species. This study reports on the inâ
vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes