title: Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.
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.
authors.affiliation: Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Lingen
authors.forename: Doris
authors.initials: D
authors.lastname: Kolter-Jung
authors.forename: Pascal
authors.initials: P
authors.lastname: Dünkelmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Feldmann
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
mesh_terms: D001665:Binding Sites; D005075:Biological Evolution; D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D007659:Ketones / Q000138:chemical synthesis*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D007659:Ketones; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase