5C71

Structure-guided engineering of the substrate specificity of a fungal beta-glucuronidase toward triterpenoid saponins.

(2018) J Biol Chem 293: 433-443

• PubMed: 29146597 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1074/jbc.M117.801910
• Primary Citation of Related Structures:  
  5C71


• 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 Aspergillus oryzae ( P GUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the P GUS structure, representing the first structure of a fungal β-glucuronidase, and that of an inactive P GUS mutant in complex with the native substrate glycyrrhetic acid 3- O -mono-β-glucuronide (GAMG). P 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 ⁴¹⁴ and Glu ⁵⁰⁵ , 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 ¹⁶² , His ³³² , Asp ⁴¹⁴ , Tyr ⁴⁶⁹ , Tyr ⁴⁷³ , Asp ⁵⁰⁵ , Arg ⁵⁶³ , Asn ⁵⁶⁷ , and Lys ⁵⁶⁹ ) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr ⁴⁶⁹ , Phe ⁴⁷⁰ , Trp ⁴⁷² , and Tyr ⁴⁷³ Finally, structure-guided mutagenesis to precisely manipulate P GUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala ³⁶⁵ and Arg ⁵⁶³ , 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.

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Organizational Affiliation: 

From the Department of Biochemical Engineering/Institute for Biotransformation and Synthetic Biosystem, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, 100081 Beijing, China and.