Improving the C_β Stereoselectivity of <scp>l</scp>‐Threonine Aldolase for the Synthesis of <scp>l‐</scp><i>threo</i>‐4‐Methylsulfonylphenylserine by Modulating the Substrate‐Binding Pocket To Control the Orientation of the Substrate Entrance

Abstract

Abstract l ‐Threonine aldolase from Actinocorallia herbida ( Ah LTA) is an ideal catalyst for producing l ‐ threo ‐4‐methylsulfonylphenylserine [(2 S ,3 R )‐ 1 b ], a key chiral precursor for florfenicol and thiamphenicol. The moderate C β stereoselectivity is the main obstacle to the industrial application of Ah LTA. To address this issue, a combinatorial active‐site saturation test (CAST) together with sequence conservatism analysis was applied to engineer the Ah LTA toward improved C β stereoselectivity. The optical mutant Y314R could asymmetrically synthesize l ‐ threo ‐4‐methylsulfonylphenylserine with 81 % diastereomeric excess ( de ), which is 23 % higher than wild‐type Ah LTA. Molecular dynamic (MD) simulations revealed that the mechanism for the improvement in C β 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 β stereoselectivity problem of LTA and contribute to the industrial application of LTA.