Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

Abstract

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

Reductive Enzyme Cascades for Valorization of Polyethylene Terephthalate Deconstruction Products

“…The formation of alcohols is not known to be catalyzed by CAR but has been reported in systems in which protein preparations may contain impurities . Recently, the ability of some CARs to also catalyze overreduction of aldehydes to alcohols was shown …”

“…69 Recently, the ability of some CARs to also catalyze overreduction of aldehydes to alcohols was shown. 70 To better understand the timescale of CAR-catalyzed reduction of PET-derived substrates, we performed a timecourse analysis of srCAR-catalyzed reduction of 5 mM TPA, 4FBA, mmTPA, and MHET (Figure 4B). After 24 h, the conversion of TPA is 77%; however, the yield of the desired dialdehyde was only 39%, with 22% of the aldehyde products overreduced to HMB.…”

Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

“…71 Recently, the ability of some CARs to also catalyze overreduction of aldehydes to alcohols was shown. 72…”

“…71 Recently, the ability of some CARs to also catalyze overreduction of aldehydes to alcohols was shown. 72 To better understand the timescale of CAR catalyzed reduction of PET-derived substrates, we performed a time-course analysis of srCAR-catalyzed reduction of 5 mM TPA, 4FBA, mmTPA, and MHET (Fig. 4B).…”

Pyridine–borane complex-catalysed thioesterification: the direct conversion of carboxylic acids to thioesters

“…Sustainably accessing valuable molecules from simple and abundant feedstocks is one of the central tasks of advanced organic synthesis. Thioesters are not only found in drug molecules, natural products, and bioactive molecules (Scheme 1a), but also present in vital synthons to ketones, 1 amides, 2 esters 3 and others, 4 as well as common intermediates in many biosynthetic reactions. 5 Classic approaches for the synthesis of thioesters mostly rely on activated acylating reagents such as highly reactive acyl chlorides and anhydrides, in the presence of activating reagents and catalysts, which emit copious waste.…”