Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

Abstract

L-Threonine transaldolase (LTTA) is an attractive biocatalyst because of its potential diastereoselectivity in the synthesis of β-hydroxy-αamino 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 (k cat /K m ) 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-pmethylsulfonylphenylserine, 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% syn de 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.