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.

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The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

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