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 fungusNeurospora
crassa(Nc). TheNcCAR
R-domain revealed activity forN-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 theNcCAR 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 vitrostudies 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-lengthNcCAR reduced aldehydes
to primary alcohols. In conclusion, aldehyde overreduction can no
longer be attributed exclusively to the host background.