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Improving and Inverting Cβ‑Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening

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journal contribution
posted on 09.04.2019, 00:00 by Qijia Chen, Xi Chen, Jinhui Feng, Qiaqing Wu, Dunming Zhu, Yanhe Ma
Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C–C bond formation under mild conditions, but the low Cβ-stereoselectivity has hampered its wide application. A stepwise visual screening method was developed to measure the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with higher Cβ-stereoselectivity. Mutants of l-PsTA from Pseudomonas sp. with improved or inverted stereoselectivity toward aromatic aldehydes were obtained by simultaneously mutating amino acid residues which interact with the amino and hydroxyl groups of the substrate and screening the resulting mutant libraries with this method. The mutation and enzyme–substrate docking studies provided some insights into the regulation of the Cβ-stereoselectivity by the enzyme–substrate interactions. This study offers a tool and useful guidance for further engineering of TAs to address the Cβ-stereoselectivity problem.

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