Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry

Abstract

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl‐CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron‐rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α‐phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP+, adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell‐Mediated Preparation of 3‐Hydroxytyrosol

“…Overall, Tv CAR showed activity in a rather broad range from pH 6.0‐8.0 without losing activity considerably (Figure A). This observation was consistent with the biophysical characterizations of other CARs . The pH optimum determined for Tv2 CAR and Ds CAR showed the highest relative activity at pH 7.5 in 100 mM bicine‐ NaOH buffer and showed a preference for neutral to basic aqueous environment (Figure B–C).…”

“…The protein sequences of Ni CAR, Mm CAR, Sro CAR and At CAR were taken as templates for a BLASTp search with the restriction to organisms with experimentally confirmed ability to reduce carboxylic acids. Hit sequences were further analyzed for the presence of essential key residues and motifs . Putative CAR sequences from Dichomitus squalens ( Ds CAR) and Trametes versicolor ( Tv2 CAR) were top rated.…”

Exploring Bacterial Carboxylate Reductases for the Reduction of Bifunctional Carboxylic Acids

“…iowensis CAR (CAR_NOCIO) is inhibited by the formation of the co-product pyrophosphate, whose hydrolysis using an inorganic pyrophosphatase (PPase) resulted in a significant increase in the reaction rate and product yield [25, 26, 43]. Therefore, we used the purified PPase from E .…”

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

“…Amidation would be of particular interest because many natural amide synthetases,s uch as NRPS [14] and ATP-grasp enzymes [15,16] ,s uffer from very narrow substrate specificity,w hich limits their applications in biocatalysis.A lternatively,h ydrolases (such as lipases [17] and proteases [18] ), which are commonly used for amide formation, [2] require systems in which very little water is present, such as organic solvents,todrive the reaction towards amide bond formation. [19][20][21][22] Four CARc andidates (CARmm from Mycobacterium marinum, [3] CARni from Nocardia iowensis, [4] CARtp from Tsukamurella paurometabola, [10] and CARse from Segniliparus rotundus, [23] )were produced recombinantly in Escherichia coli,w ith the coexpressed gene for the Bacillus subtilis phosphopantetheinyl transferase (PPTase;S fp), [24] which is required for post-translational addition of the PPant group. [4] These CARs were purified (Supporting Information, Figure S1) and the amidation of carboxylic acid substrates 1-6 was tested.…”

“…CARs are composed of three distinct protein domains:t he adenylation domain, which itself is divided into ac ore-domain and subdomain, [7] the peptidyl carrier protein with the bound 4'-phosphopantetheine (PPant) prosthetic group,and areductase domain. [3] CARs have ab road substrate range and an umber of examples from different organisms are now available,m aking them attractive for applications in singlestep biotransformations, [9,10] as well as in enzymatic cascades. [8] Thea ctivated substrate is then attacked by the thiol of the enzyme-bound PPant group,generating athioester intermediate that is transferred to the reduction domain.…”