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

Graphical CASTing call : A semi-rational combinatorial active-site saturation test (CAST) was performed to improve the C β stereoselectivity of threonine aldolase. MD simulations suggested that the Y314R variant could interact with 4-methylsulfonyl benzaldehyde through hydrogen bonding to control the direction of entry of substrate, this increased the de value from 58 to 81 %. 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.