5C71

The structure of Aspergillus oryzae a-glucuronidase complexed with glycyrrhetinic acid monoglucuronide

5C71 の概要

• エントリーDOI: 10.2210/pdb5c71/pdb
• 関連するPDBエントリー: 5c70
• 分子名称: Glucuronidase, (3BETA,5BETA,14BETA)-3-HYDROXY-11-OXOOLEAN-12-EN-29-OIC ACID, alpha-D-glucopyranuronic acid, ... (4 entities in total)
• 機能のキーワード: beta-glucuronidase; glycyrrhetinic acid monoglucuronide, hydrolase
• 由来する生物種: Aspergillus oryzae
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 286311.08

構造登録者

Sun, H.L.,Lv, B.,Huang, S.,Li, C.,Jiang, T. (登録日: 2015-06-24, 公開日: 2016-06-29, 最終更新日: 2023-11-29)

主引用文献

Lv, B.,Sun, H.,Huang, S.,Feng, X.,Jiang, T.,Li, C.
Structure-guided engineering of the substrate specificity of a fungal beta-glucuronidase toward triterpenoid saponins.
J.Biol.Chem., 293:433-443, 2018

PubMed Abstract: 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 (GUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the GUS structure, representing the first structure of a fungal β-glucuronidase, and that of an inactive GUS mutant in complex with the native substrate glycyrrhetic acid 3--mono-β-glucuronide (GAMG). GUS 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, Glu and Glu, 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 (Asp, His, Asp, Tyr, Tyr, Asp, Arg, Asn, and Lys) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr, Phe, Trp, and Tyr Finally, structure-guided mutagenesis to precisely manipulate GUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala and Arg, 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.

PubMed: 29146597
DOI: 10.1074/jbc.M117.801910

実験手法

X-RAY DIFFRACTION (2.62 Å)