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

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.