Enhancing Diastereoselectivity of l‑Threonine Aldolase by Strengthening Kinetic Control To Mitigate Thermodynamic...

The reaction catalyzed by l-threonine aldolase (LTA) for the formation of β-hydroxy-α-amino acids is governed by both kinetic and thermodynamic control, causing the diastereoselectivity of LTA to gradually decrease as the reaction progresses. This hinders the enzyme's industrial application. To address this, we proposed a strategy to enhance kinetic control via substrate tunnel engineering, thereby mitigating the influence of thermodynamic equilibrium. We investigated hotspot amino acid residues in the substrate tunnel using the VR-3M mutant from Bacillus nealsonii. As a result, we obtained mutant N6A/Q303K, which exhibited a diastereoselectivity of up to 99.0% at maximum conversion for the synthesis of (2S,3R)-3-[4-(methylsulfonyl)-phenylserine]. The kinetic control in this mutant was significantly enhanced, maintaining a diastereomeric excess (de) value above 95% until 500 mina 340 min delay over the original VR-3M, which drops below 95% after 160 min while reaching only a 40.9% de value at 500 min. Molecular dynamics simulations suggest that increases in active center volume and substrate tunnel reshaping are key factors enhancing the kinetic control of diastereoselectivity. This study offers valuable insights into the interplay between kinetic and thermodynamic control in enzymatic catalysis.