Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins
| Autor: | Xudong Feng, Hanli Sun, Shen Huang, Bo Lv, Tao Jiang, Chun Li |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Glycan Glycoside Hydrolases Stereochemistry Protein Engineering 01 natural sciences Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound TIM barrel Moiety Glycoside hydrolase Molecular Biology Glucuronidase chemistry.chemical_classification biology 010405 organic chemistry Chemistry Substrate (chemistry) Cell Biology Saponins Triterpenes 0104 chemical sciences Amino acid 030104 developmental biology Aglycone Enzymology biology.protein Glycyrrhetinic Acid Homotetramer |
| Zdroj: | Journal of Biological Chemistry. 293:433-443 |
| ISSN: | 0021-9258 |
| Popis: | Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal β-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal β-glucuronidase, and that of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-β-glucuronide (GAMG). PGUS displayed a homotetramer structure with each monomer comprising three distinct domains: a sugar-binding, an immunoglobulin-like β-sandwich, and a TIM barrel domain. Two catalytic residues, Glu414 and Glu505, acted as acid/base and nucleophile, respectively. Structural and mutational analyses indicated that the GAMG glycan moiety is recognized by polar interactions with nine residues (Asp162, His332, Asp414, Tyr469, Tyr473, Asp505, Arg563, Asn567, and Lys569) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr469, Phe470, Trp472, and Tyr473. Finally, structure-guided mutagenesis to precisely manipulate PGUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala365 and Arg563, are critical for substrate specificity. Moreover, we obtained several mutants with dramatically improved GAMG yield (>95%). Structural analysis suggested that modulating the interaction of β-glucuronidase simultaneously toward glycan and aglycone moieties is critical for tuning its substrate specificity toward triterpenoid saponins. |
| Databáze: | OpenAIRE |
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