phenylserine and florfenicol

l-Threonine aldolases (l-TAs) catalyze the aldol condensation of aldehyde and glycine, offering direct enzymatic synthesis of β-hydroxy-α-amino acids under mild conditions. However, this method suffers from moderate yield and low stereoselectivity at the β-carbon. Given the importance of 4-(methylsulfonyl)phenylserine for the synthesis of florfenicol and thiamphenicol, mutations of a l-threonine aldolase from Pseudomonas sp. (l-PsTA) were performed in this study by error-prone PCR and combinatorial mutation. Some beneficial mutants were obtained by screening the mutant library using a stepwise visual method. 4-(Methylsulfonyl)phenylserine was synthesized in up to 71 % diastereomeric excess (de), which are much higher than the previously reported 2 % de value, by using the newly identified mutants. The mutants V200I and C187S/V200I were found to improve the product yield and stereoselectivity for the aldol condensation of various benzaldehydes with glycine. These results show that the amino acid residues outside of the substrate-binding cavity of l-PsTA play an important role in determining its C

Topics: Catalysis; Glycine Hydroxymethyltransferase; Mutation; Protein Engineering; Pseudomonas; Serine; Substrate Specificity; Thiamphenicol