Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis

Abstract

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the non-ribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that reduction of the aldehyde product does not occur. Combining crystallography with small-angle x-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

“…[7] This construct lacks the terminal reduction domain but retains the adenylation domain and carrier protein (CARmmD729-1175;F igure 2A). [7] This construct lacks the terminal reduction domain but retains the adenylation domain and carrier protein (CARmmD729-1175;F igure 2A).…”

“…These variants might have application as amidation catalysts in more complex enzyme cascades,b oth in cell-free and whole-cell systems,w here the presence of NADPH would normally suppress CAR-mediated amidation in favor of reduction. [7]). Ther eaction was tolerant to ar ange of carboxylic acids and amines and could be performed in an aqueous medium using ATPa st he driving force.T he method was applied to at arget drug molecule, ilepcimide 20,w ith up to 96 %c onversion and preparative scale-up.G iven that all four CARs tested exhibited this activity,t he range of enzymes that could be used for this amidation reaction might be rapidly expanded by tapping into the collection of known CARe nzymes with differing and broad substrate specificities.…”

“…[2] An interesting group are carboxylic acid reductases (CAR), [3,4] which catalyze the reduction of carboxylic acids to aldehydes via acyl adenylates and thioesters ( Figure 1). [6] Our recent structural work [7] has shown that CARs are particularly good candidates for this approach. Of specific interest were amine nucleophiles that would lead to amide bond formation from acid and amine,an important challenge in organic chemistry.…”

“…Given that both activated esters are shared with other enzyme pathways,s uch as those of nonribosomal peptide synthetases (NRPS), [5] we were interested in exploring the activity of CARs towards other nucleophiles ( Figure 1). 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. [6] Our recent structural work [7] has shown that CARs are particularly good candidates for this approach.…”

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

“…Their 3‐domain‐architecture as well as the reaction scheme are displayed in Figure . Their ATP and NADPH dependent mechanism was affirmed by the determination of protein crystal structures of separate adenylation and reductase domains of bacterial CARs and modelling . CARs are frequently introduced into cascade reactions in vivo .…”