Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors

Saino, Hiromichi; Shimizu, Tetsuya; Hiratake, Jun; Nakatsu, Toru; Kato, Hiroaki; Sakata, Kanzo; Mizutani, Masaharu

doi:10.1074/jbc.m114.553271

Cited by 16 publications

(8 citation statements)

References 48 publications

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“…The extensive polar interaction network between the enzyme and the glycan may explain the strict specificity of PGUS toward the glycan moiety ( Fig. 3) in agreement with most GHs from different families in which rigorous specificity toward glycan has been reported (25,29).…”

Section: The Structure Of Pgus Complexed With Gamgsupporting

confidence: 82%

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

Sun

Huang

et al. 2018

Journal of Biological Chemistry

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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 inactiveGUS 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.

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Section: The Structure Of Pgus Complexed With Gamgsupporting

confidence: 82%

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

Sun

Huang

et al. 2018

Journal of Biological Chemistry

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“…Significantly more information, including one tertiary [18] and several primary structures, is available on primeverosidases. These enzymes constitute a different class of diglycosidases, cleaving the glycosidic linkage between the aglycone and b-prime-…”

Section: Expression and Purification Of Recombinant Rutinosidasementioning

confidence: 99%

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

et al. 2014

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Abstract:We report the first heterologous production of a fungal rutinosidase (6-O-a-l-rhamnopyranosyl-b-d-glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the a-l-rhamnosidase-catalyzed synthesis of a chromogenic substrate for a rutinosidase assay -para-nitrophenyl b-rutinoside -is described.

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“…Several codon randomization schemes [15] , [16] can be used to generate variants of proteins with different sets of amino acids at selected positions. For random saturation mutagenesis (A/C/G/T, A/C/G/T, G/T) or NNN (A/C/G/T, A/C/G/T, A/C/G/T) codon randomization are widely applied to introduce all amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…Here the hydrophobic cluster F193–F200–W373–F461 in Zm-p60.1 corresponds to G210-L217-A387-L472 in β-primeverosidase. The unique combination of small amino acid residues, and most importantly the absence of tryptophan at the 387 position, forms a large cavity providing the basis for the broad aglycone specificity of β-primeverosidase and possibility to accept aglycones with a bulky disaccharide-glycone part [16] .…”

Section: Resultsmentioning

confidence: 99%

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

et al. 2014

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Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins’ structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations’ effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

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Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors

Cited by 16 publications

References 48 publications

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

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

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

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