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<p>In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (<i>R</i>)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (<i>R</i>)-phenylethylamine ((<i>R</i>)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (<i>R</i>)-ATA.</p>

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The pedal-like loop of (R)-selective transaminases plays a critical role to the functionality of the enzyme

  • 期刊论文

  • 发表日期：

    2025年3月18日

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  • Chao Xiang,
  • Yu-Ke Ce,
  • Ya-Ping Xue,
  • Yu-Guo Zheng

摘要

In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (R)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (R)-phenylethylamine ((R)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (R)-ATA.