Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.

High-throughput assay of ( R)-phenylacetylcarbinol synthesized by pyruvate decarboxylase.

A novel assay has been developed for the detection of ( R)-phenylacetylcarbinol, ( R)-PAC, a chiral intermediate in the industrial synthesis of ephedrine. It is the product of a biotransformation of benzaldehyde catalysed by the enzyme pyruvate decarboxylase. The assay, using 2,3,5-triphenyltetrazolium chloride, enables high-throughput photometric analysis of the activity of the enzyme thus avoiding time-consuming chromatographic procedures.

Thiamin-diphosphate-dependent enzymes: new aspects of asymmetric C-C bond formation.

Starting from a thorough investigation of mechanistic aspects of ThDP-dependent (ThDP = thiamin diphosphate) enzymes in combination with mutagenesis studies and a detailed substrate screening, new general synthetic methods have been developed based on Umpolung reactions by thiamin catalysis. A selective donor-acceptor concept was established leading to the first asymmetric cross-benzoin condensation, and a kinetic racemic resolution through C-C bond cleavage was developed. With these tools and in combination with protein engineering, we approached the synthesis of new chiral building blocks on a preparative scale. An outlook is given with respect to the potential of other ThDP-dependent enzymes as catalysts in asymmetric synthesis.

Development of a donor-acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation.

Highly enantioenriched mixed benzoins are obtained selectively through a biocatalytical cross-coupling reaction of aromatic aldehydes using ThDP-dependent enzymes.