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Literature summary for 3.2.1.31 extracted from
- Structure-guided engineering of the substrate specificity of a fungal beta-glucuronidase toward triterpenoid saponins (2018), J. Biol. Chem., 293, 433-443 .
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | mutant enzyme A365H/R563E has great potential in the industrial production of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of the protein His6-tagged at its N terminus in Escherichia coli | Aspergillus oryzae |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| sitting drop vapor diffusion method at 16°C, structure of the enzyme is determined at 3.1 A resolution and structure of the inactive mutant enzyme E414D/E505D in complex with the native substrate glycyrrhetic acid 3-O-mono-beta-glucuronide at 2.6 A resolution | Aspergillus oryzae |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| A365H/R563E | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 96% compared to less than 10% produced by wild-type enzyme, resulting in nearly complete alteration of the substrate selectivity of the glucuronyl hydrolase from glycyrrhetic acid to glycyrrhetinic acid 3-O-mono-beta-D-glucuronide formation. No activity with glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae |
| A365Q | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 65% compared to less than 10% produced by wild-type enzyme | Aspergillus oryzae |
| A365T | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 61% compared to less than 10% produced by wild-type enzyme | Aspergillus oryzae |
| A365T/R563E | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 95% compared to less than 10% produced by wild-type enzyme, resulting in nearly complete alteration of the substrate selectivity of the glucuronyl hydrolase from glycyrrhetic acid to glycyrrhetinic acid 3-O-mono-beta-D-glucuronide formation. No activity with glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae |
| E414A | inactive mutant enzyme | Aspergillus oryzae |
| E414D/E505D | inactive mutant enzyme | Aspergillus oryzae |
| E505A | inactive mutant enzyme | Aspergillus oryzae |
| R563E | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 77% compared to less than 10% produced by wild-type enzyme | Aspergillus oryzae |
| R563K | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 58% compared to less than 10% produced by wild-type enzyme. The catalytic efficiency (kcat/Km) toward glycyrrhetinic acid 3-O-mono-beta-D-glucuronide decreases by 88.4%, whereas kcat/Km toward GL decreases by 12.3%, confirming site 563 has a dramatic effect on substrate specificity | Aspergillus oryzae |
| R563Q | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 42% compared to less than 10% produced by wild-type enzyme | Aspergillus oryzae |
| V447Q | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 81% compared to less than 10% produced by wild-type enzyme | Aspergillus oryzae |
| V447Q/R563K | the yield of glycyrrhetinic acid 3-O-mono-beta-D-glucuronide of the mutant enzyme is 95% compared to less than 10% produced by wild-type enzyme, resulting in nearly complete alteration of the substrate selectivity of the glucuronyl hydrolase from glycyrrhetic acid to glycyrrhetinic acid 3-O-mono-beta-D-glucuronide formation. No activity with glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.78 | - |
glycyrrhizin | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae |  |
| 0.91 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 0.92 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae | |
| 0.97 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 1.06 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 1.1 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 1.21 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 1.23 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 1.62 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q/R563K | Aspergillus oryzae | |
| 2.2 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T/R563E | Aspergillus oryzae | |
| 2.68 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365H/R563E | Aspergillus oryzae | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 290000 | - |
ultracentrifugation | Aspergillus oryzae |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Aspergillus oryzae | A7XS03 | - |
- |
| Aspergillus oryzae Li-3 | A7XS03 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| Ni2+-affinity chromatography, anion-exchange chromatography, and gel filtration | Aspergillus oryzae |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 4-nitrophenol beta-D-glucuronide + H2O | - |
Aspergillus oryzae | 4-nitrophenol + beta-D-glucuronate | - |
? | |
| 4-nitrophenol beta-D-glucuronide + H2O | - |
Aspergillus oryzae Li-3 | 4-nitrophenol + beta-D-glucuronate | - |
? | |
| glycyrrhetinic acid 3-O-mono-beta-D-glucuronide + H2O | the enzyme shows similar activities toward both glycyrrhizin and glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae | glycyrrhetic acid + D-glucuronate | - |
? | |
| glycyrrhetinic acid 3-O-mono-beta-D-glucuronide + H2O | the enzyme shows similar activities toward both glycyrrhizin and glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae Li-3 | glycyrrhetic acid + D-glucuronate | - |
? | |
| glycyrrhizin + H2O | the enzyme shows similar activities toward both glycyrrhizin and glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae | glycyrrhetinic acid 3-O-mono-beta-D-glucuronide + D-glucuronate | - |
? | |
| glycyrrhizin + H2O | the enzyme shows similar activities toward both glycyrrhizin and glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | Aspergillus oryzae Li-3 | glycyrrhetinic acid 3-O-mono-beta-D-glucuronide + D-glucuronate | - |
? | |
| additional information | the enzyme does not show strict specificity toward the aglycone moiety and exhibits activity toward all the natural and artificial substrates. Strict glycan specificity and hydrolyzes only the artificial substrate containing glucuronide groups | Aspergillus oryzae | ? | - |
? | |
| additional information | the enzyme does not show strict specificity toward the aglycone moiety and exhibits activity toward all the natural and artificial substrates. Strict glycan specificity and hydrolyzes only the artificial substrate containing glucuronide groups | Aspergillus oryzae Li-3 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homotetramer | each monomer comprises three distinct domains: a sugar-binding, an immunoglobulin-like beta-sandwich, and a TIM barrel domain | Aspergillus oryzae |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PGUS | - |
Aspergillus oryzae |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | - |
assay at | Aspergillus oryzae |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.89 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 2.72 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T/R563E | Aspergillus oryzae | |
| 3.06 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365H/R563E | Aspergillus oryzae | |
| 3.23 | - |
glycyrrhizin | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae | |
| 3.28 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 3.32 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 3.52 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 4.36 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 6.03 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 7.01 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q/R563K | Aspergillus oryzae | |
| 7.81 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 5 | - |
assay at | Aspergillus oryzae |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.98 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 1.14 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365H/R563E | Aspergillus oryzae | |
| 1.23 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T/R563E | Aspergillus oryzae | |
| 2.67 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 3.06 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme A365T | Aspergillus oryzae | |
| 3.13 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 3.63 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme R563K | Aspergillus oryzae | |
| 4.14 | - |
glycyrrhizin | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae | |
| 4.32 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q/R563K | Aspergillus oryzae | |
| 4.98 | - |
glycyrrhizin | pH 5.0, 40°C, mutant enzyme V447Q | Aspergillus oryzae | |
| 8.49 | - |
glycyrrhetinic acid 3-O-mono-beta-D-glucuronide | pH 5.0, 40°C, wild-type enzyme | Aspergillus oryzae | |
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