Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (PcCAR2) was expressed in Escherichia coli. PcCAR2's biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. PcCAR2 was immobilized to explore its potential as a recyclable biocatalyst. PcCAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst.
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.