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Improving the C β 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
Author(s) -
Wang Lichao,
Xu Lian,
Su Bingmei,
Lin Wei,
Xu Xinqi,
Lin Juan
Publication year - 2021
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.202100752
Subject(s) - stereoselectivity , chemistry , stereochemistry , aldolase a , diastereomer , active site , enantioselective synthesis , combinatorial chemistry , biochemistry , catalysis , enzyme
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.