Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution

✍ Scribed by Bettina Lingen; Doris Kolter-Jung; Pascal Dünkelmann; Ralf Feldmann; Joachim Grötzinger; Martina Pohl; Michael Müller

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 136 KB
• Volume: 4
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/cbic.200200475

✦ Synopsis

Abstract

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.__