Improving the Cβ Stereoselectivity of L-Threonine Aldolase for the Synthesis of L-threo-4-Methylsulfonylphenylserine by Modulating the Substrate-Binding Pocket To Control the Orientation of the Substrate Entrance

l-Threonine aldolase from Actinocorallia herbida (AhLTA) is an ideal catalyst for producing l-threo-4-methylsulfonylphenylserine [(2S,3R)-1 b], a key chiral precursor for florfenicol and thiamphenicol. The moderate C-beta stereoselectivity is the main obstacle to the industrial application of AhLTA. To address this issue, a combinatorial active-site saturation test (CAST) together with sequence conservatism analysis was applied to engineer the AhLTA toward improved C-beta stereoselectivity. The optical mutant Y314R could asymmetrically synthesize l-threo-4-methylsulfonylphenylserine with 81 % diastereomeric excess (de), which is 23 % higher than wild-type AhLTA. Molecular dynamic (MD) simulations revealed that the mechanism for the improvement in C-beta stereoselectivity of Y314R is due to the acylamino group of residues Arg314 controlling the orientation of substrate 4-methylsulfonyl benzaldehyde (1 a) in the active pocket by directed interaction with the methylsulfonyl group; this leads to asymmetric synthesis of l-threo-4-methylsulfonylphenylserine. The success in this study demonstrates that direct control of substrates in an active pocket is an attract strategy to address the C-beta stereoselectivity problem of LTA and contribute to the industrial application of LTA.

Automated summary

l-Threonine aldolase from Actinocorallia herbida (AhLTA) is an ideal catalyst for producing l-threo-4-methylsulfonylphenylserine, and engineering the enzyme through a combinatorial active-site saturation test (CAST) and sequence conservatism analysis has successfully improved the C-beta stereoselectivity. The mutant Y314R demonstrated higher diastereomeric excess (de) in asymmetric synthesis, showcasing the potential of directly controlling substrates in the active pocket for industrial applications.