Zhiwen Xi, Jingxin Rao, Xinyi Zhang, Zhiyong Liu, Mingyue Zheng, Lihong Li, Wenchi Zhang, Yan Xu and Rongzhen Zhang*,
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引用次数: 0
Abstract
l-Threonine transaldolase (LTTA) is an attractive biocatalyst because of its potential diastereoselectivity in the synthesis of β-hydroxy-α-amino acids (βHAAs). However, prospective development of LTTA has been hampered by its low activity. Here, a combination of techniques involving structural comparison, computational analysis, Loop deletion, and alanine scanning was used to identify a key Loop region (Loop 1) regulating the catalytic ability of Chitiniphilus shinanonensis LTTA (CsLTTA). Saturation mutagenesis and iterative saturation mutagenesis at the hot spots in Loop 1 were performed, and the best variant containing an F70T/C57Q/Y69T (TQT) triple mutation was screened. The diastereoisomer excess (de) produced by the TQT variant (95.4%syn) was greater than that produced by the wild-type (WT) enzyme (75.2%syn), and the catalytic efficiency (kcat/Km) of the TQT variant was four times higher than that of the wild-type enzyme. Molecular dynamics simulations, metadynamics simulations, and CAVER analysis revealed the critical role of the Loop 1 structure in regulating the hydrogen bond network and thus reshaping the active-site pocket to control the syn-tunnel direction. Further engineering of Loop 1 in ObiH, an LTTA responsible for obafluorin biosynthesis, resulted in the development of the F70T-C57Q-H69T (ObiH-TQT) variant producing a de of 97%syn. Using the ObiH-TQT variant for kilogram-scale synthesis of l-syn-p-methylsulfonylphenylserine, coupled with acetaldehyde elimination, resulted in space–time yields of up to 12.7 g L–1 h–1. The method achieved 98.3% substrate conversion and 99.2%synde within 6 h, marking the highest reported levels to date. The above findings will contribute to the industrial production of β-hydroxy-α-amino acids, offer insights into the mechanism of Loop regions regulating the catalytic function of LTTAs, and provide ideas for engineering other enzymes.
期刊介绍:
ACS Catalysis is an esteemed journal that publishes original research in the fields of heterogeneous catalysis, molecular catalysis, and biocatalysis. It offers broad coverage across diverse areas such as life sciences, organometallics and synthesis, photochemistry and electrochemistry, drug discovery and synthesis, materials science, environmental protection, polymer discovery and synthesis, and energy and fuels.
The scope of the journal is to showcase innovative work in various aspects of catalysis. This includes new reactions and novel synthetic approaches utilizing known catalysts, the discovery or modification of new catalysts, elucidation of catalytic mechanisms through cutting-edge investigations, practical enhancements of existing processes, as well as conceptual advances in the field. Contributions to ACS Catalysis can encompass both experimental and theoretical research focused on catalytic molecules, macromolecules, and materials that exhibit catalytic turnover.