Objectives: To develop robust variants of L-threonine aldolases (L-TAs), potent catalysts for synthesizing asymmetric β-hydroxy-α-amino acids, it is necessary to identify critical residues beyond the known active site residues.
Results: Through virtual screening, a neglected residue Asn305, was identified as critical for catalytic efficiency. Subsequent site-saturation mutagenesis led to a potent variant N305R which exhibited excellent conversions of 88%conv (87%de) and 80%conv (94%de) for the synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine respectively. This variant not only outperformed the template enzyme, but also represented a promising L-TA for synthesizing the two β-hydroxy-α-amino acids. It was suggested that Arg305 of the variant N305R generated strong cation-arene interaction and electrostatic force with the intermediates, leading to strengthened binding, enhanced L-threo favored orientation and wider entrance.
Conclusions: Our work not only provided an excellent variant N305R, but also suggested the crucial function of a neglected residue Asn305, which offered valuable experiences for other L-TA studies.
Keywords: β-Hydroxy-α-amino acids; Diastereoselectivity; Enzymatic synthesis; L-Threonine aldolase.
© 2024. The Author(s), under exclusive licence to Springer Nature B.V.