TY - JOUR
T1 - Enhancing the Catalytic Specificity of the β-Glucuronidase AtGUS from Aspergillus terreus Li-20 by Site-Directed Mutagenesis on Loop 8
AU - Liu, Yanli
AU - Li, Xinying
AU - Cheng, Ruichen
AU - Wang, Chao
AU - Dai, Dazhang
AU - Li, Chun
N1 - Publisher Copyright:
© 2025 American Chemical Society.
PY - 2025
Y1 - 2025
N2 - AtGUS, identified in the genome of Aspergillus terreus Li-20, can hydrolyze glycyrrhetinic (GL) into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). However, the poor substrate specificity of AtGUS often limits its further application. In this study, the highly conserved Glu416 and Glu507 residues were identified as the catalytic sites of AtGUS, while Glu158, Asp163, and Arg565, which form hydrogen bonds with the substrate, play critical roles in the enzymatic activity, with Arg565 being particularly important. When Arg565 on loop 8 of the surface of the TIM barrel was mutated to glutamate, GAMG became the sole catalytic product, and the yield of GAMG from GL conversion was up to 85%. When the hydrolysis reaction was terminated, the yield of GAMG was 7.32-fold higher than that of the wild enzyme. Molecular docking and dynamic simulation analyses revealed that the affinity for the mutant R565E (Km = 0.192 mmol/L) to the substrate GL was improved, but with reduced catalytic efficiency toward GAMG (kcat = 5.515 ± 0.07 mmol-1 L s-1). This study provides new insights into understanding the catalytic mechanism and new ideas for efficient enzymatic preparation of GAMG.
AB - AtGUS, identified in the genome of Aspergillus terreus Li-20, can hydrolyze glycyrrhetinic (GL) into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). However, the poor substrate specificity of AtGUS often limits its further application. In this study, the highly conserved Glu416 and Glu507 residues were identified as the catalytic sites of AtGUS, while Glu158, Asp163, and Arg565, which form hydrogen bonds with the substrate, play critical roles in the enzymatic activity, with Arg565 being particularly important. When Arg565 on loop 8 of the surface of the TIM barrel was mutated to glutamate, GAMG became the sole catalytic product, and the yield of GAMG from GL conversion was up to 85%. When the hydrolysis reaction was terminated, the yield of GAMG was 7.32-fold higher than that of the wild enzyme. Molecular docking and dynamic simulation analyses revealed that the affinity for the mutant R565E (Km = 0.192 mmol/L) to the substrate GL was improved, but with reduced catalytic efficiency toward GAMG (kcat = 5.515 ± 0.07 mmol-1 L s-1). This study provides new insights into understanding the catalytic mechanism and new ideas for efficient enzymatic preparation of GAMG.
KW - glycyrrhetinic acid monoglucuronide (GAMG)
KW - glycyrrhizin (GL)
KW - loop
KW - substrate specificity
KW - β-glucuronidase
UR - http://www.scopus.com/inward/record.url?scp=85214571542&partnerID=8YFLogxK
U2 - 10.1021/acsagscitech.4c00477
DO - 10.1021/acsagscitech.4c00477
M3 - Article
AN - SCOPUS:85214571542
SN - 2692-1952
JO - ACS Agricultural Science and Technology
JF - ACS Agricultural Science and Technology
ER -