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.
Lingen
Bettina
B
Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany.
Kolter-Jung
Doris
D
Dünkelmann
Pascal
P
Feldmann
Ralf
R
Grötzinger
Joachim
J
Pohl
Martina
M
Müller
Michael
M
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
D007659:Ketones; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase