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

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 (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.

Keywords: crystal structure; enzyme mutation; fungal β-glucuronidase; glycoside hydrolase; protein engineering; substrate specificity; triterpenoid saponins.

Figure 1.

Nonspecific hydrolysis of GL to GAMG and GA by GUSs (wild type) without selectivity due to the low specificity toward aglycone moieties.

Figure 2.

a, crystal structure of PGUS (asymmetric unit). b, structure of one monomer of PGUS (the sugar-binding domain is green, the TIM barrel domain is cyan, and the immunoglobulin-like β-sandwich domain is magenta). The two catalytic sites, Glu⁴¹⁴ and Glu⁵⁰⁵, are shown as red sticks. c, overview of the active site of PGUS. d, ribbon diagram of the six loops that form the substrate entry to the active site of PGUS: loop A (red), loop B (blue), loop C (magenta), loop D (orange), loop E (yellow), and loop F (cyan).

Figure 3.

Substrate recognition and binding of PGUS elucidated from the crystal structure of PGUS complexed with GAMG. a, the active site pocket of PGUS in complex with GAMG. The electron density for GAMG is also shown. b, analysis of the interactions between GAMG and PGUS. GAMG is shown as blue sticks, and the residues of PGUS that interact with the glycan are shown as green sticks. c, a schematic diagram of the detailed interaction network drawn on the basis of the structure in b.

Figure 4.

a, ribbon diagram of loop D (361–373) and GAMG. Loop D (361–373) is not well-defined due to its weak electron density. b, loop D (361–373) was reconstructed using SWISS-MODEL, and its location suggests that it is involved in aglycone recognition. The structure of PGUS is shown as green schematics, and GAMG is shown as blue sticks.

Figure 5.

a, the effects of the saturated mutagenesis of Arg⁵⁶³ on the substrate specificity of PGUS. b, the effects of the combination of mutations of Ala³⁶⁵, Val⁴⁴⁷, and Arg⁵⁶³ on the substrate specificity of PGUS. Error bars represent S.D.

Figure 6.

Time course of the hydrolysis of GL by mutants A365H/R563E and V447Q/R563K.

Figure 7.

a, the superimposition of structures of PGUS and mutants R563Q, R563K, and R563E docked with GAMG. Only critical residues are displayed for clarity. The mutagenesis was performed using PyMOL, and the structure was optimized by VMD (Visual Molecular Dynamics) software with energy minimization. b, the active site pocket of PGUS in complexed with GAMG (from crystal structure) and GL (from docking). The docking was performed with Autodock software.