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Literature summary extracted from
- Functional characterization of recombinant hyoscyamine 6beta-hydroxylase from Atropa belladonna (2012), Bioorg. Med. Chem., 20, 4356-4363.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.14.11.11 | DNA and amino acid sequence determination and analysis, overexpression of N- or C-terminally His-tagged enzyme in Escherichia coli strain BL21 (DE3), AbH6H loses of the first amino acid methionine during transcription | Atropa belladonna |
| 1.14.20.13 | DNA and amino acid sequence determination and analysis, overexpression of N- or C-terminally His-tagged enzyme in Escherichia coli strain BL21 (DE3), AbH6H loses of the first amino acid methionine during transcription | Atropa belladonna |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.11.11 | (1R,2R,4S,5S,7s)-7-{[(2S)-3-hydroxy-2-phenylpropanoyl]oxy}-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.02,4]nonane | slight inhibition by the substrate analogue | Atropa belladonna |  |
| 1.14.11.11 | 1-methylpiperidin-4-yl 2-phenylacetate | - |
Atropa belladonna | |
| 1.14.11.11 | 6,7-Dehydrohyoscyamine | - |
Atropa belladonna | |
| 1.14.11.11 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate | - |
Atropa belladonna | |
| 1.14.11.11 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate | - |
Atropa belladonna | |
| 1.14.11.11 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl benzoate | - |
Atropa belladonna | |
| 1.14.11.11 | 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate | - |
Atropa belladonna | |
| 1.14.11.11 | additional information | synthesis and inhibitory potencies of substrate analogues, overview. No inhibition by substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, and 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate | Atropa belladonna | |
| 1.14.20.13 | (1R,2R,4S,5S,7s)-7-{[(2S)-3-hydroxy-2-phenylpropanoyl]oxy}-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.02,4]nonane | slight inhibition by the substrate analogue | Atropa belladonna | |
| 1.14.20.13 | 1-methylpiperidin-4-yl 2-phenylacetate | - |
Atropa belladonna | |
| 1.14.20.13 | 6,7-Dehydrohyoscyamine | - |
Atropa belladonna | |
| 1.14.20.13 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate | - |
Atropa belladonna | |
| 1.14.20.13 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate | - |
Atropa belladonna | |
| 1.14.20.13 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl benzoate | - |
Atropa belladonna | |
| 1.14.20.13 | 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate | - |
Atropa belladonna | |
| 1.14.20.13 | additional information | synthesis and inhibitory potencies of substrate analogues, overview. No inhibition by substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, and 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate | Atropa belladonna |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.11.11 | 0.0521 | - |
L-hyoscyamine | pH 7.6, 34°C, recombinant enzyme | Atropa belladonna | |
| 1.14.20.13 | 0.0844 | - |
(6S)-6beta-hydroxyhyoscyamine | pH 7.6, 34°C, recombinant enzyme | Atropa belladonna | |
Molecular Weight [Da]
| EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|---|
| 1.14.11.11 | 39238 | - |
1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
| 1.14.11.11 | 39369 | - |
1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
| 1.14.20.13 | 39238 | - |
1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
| 1.14.20.13 | 39369 | - |
1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.11.11 | L-hyoscyamine + 2-oxoglutarate + O2 | Atropa belladonna | - |
(6S)-hydroxyhyoscyamine + succinate + CO2 | - |
? | |
| 1.14.11.11 | additional information | Atropa belladonna | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine | ? | - |
? | |
| 1.14.20.13 | (6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 | Atropa belladonna | via 6,7-dehydrohyoscyamine or not, two possible pathways for the epoxidation in the biosynthesis of scopolamine, overview | scopolamine + succinate + CO2 | - |
? | |
| 1.14.20.13 | additional information | Atropa belladonna | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps, the enzyme also catalyzes the reaaction of EC 1.14.11.11. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine | ? | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.14.11.11 | Atropa belladonna | - |
- |
- |
| 1.14.20.13 | Atropa belladonna | - |
- |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 1.14.11.11 | recombinant N- or C-terminally His-tagged from Escherichia coli by nickel affinity chromatography | Atropa belladonna |
| 1.14.20.13 | recombinant N- or C-terminally His-tagged from Escherichia coli by nickel affinity chromatography | Atropa belladonna |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 1.14.11.11 | L-hyoscyamine + 2-oxoglutarate + O2 = (6S)-hydroxyhyoscyamine + succinate + CO2 | proposed mechanism of epoxidation catalyzed by the enzyme, overview | Atropa belladonna | |
| 1.14.20.13 | (6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 = scopolamine + succinate + CO2 + H2O | proposed mechanism of epoxidation catalyzed by the enzyme, overview | Atropa belladonna |
Storage Stability
| EC Number | Storage Stability | Organism |
|---|---|---|
| 1.14.11.11 | -20°C, purified recombinant enzyme, 20 mM phosphate, 2 months, loss of approximately 20% of activity | Atropa belladonna |
| 1.14.11.11 | 4°C, purified recombinant enzyme, 20 mM phosphate, 4 days, loss of over 50% activity | Atropa belladonna |
| 1.14.20.13 | -20°C, purified recombinant enzyme, 20 mM phosphate, 2 months, loss of approximately 20% of activity | Atropa belladonna |
| 1.14.20.13 | 4°C, purified recombinant enzyme, 20 mM phosphate, 4 days, loss of over 50% activity | Atropa belladonna |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.11.11 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate + 2-oxoglutarate + O2 | - |
Atropa belladonna | ? | - |
? | |
| 1.14.11.11 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl benzoate + 2-oxoglutarate + O2 | - |
Atropa belladonna | ? | - |
? | |
| 1.14.11.11 | L-hyoscyamine + 2-oxoglutarate + O2 | - |
Atropa belladonna | (6S)-hydroxyhyoscyamine + succinate + CO2 | - |
? | |
| 1.14.11.11 | additional information | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine | Atropa belladonna | ? | - |
? | |
| 1.14.11.11 | additional information | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. The catalytic efficiency of AbH6H, especially for the second oxidation, which is the reaction of EC 1.14.11.14, is low. The epoxidation step is much slower than the hydroxylation step. Substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate, and 4 are no substrates for the enzyme | Atropa belladonna | ? | - |
? | |
| 1.14.20.13 | (6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 | - |
Atropa belladonna | scopolamine + succinate + CO2 | - |
? | |
| 1.14.20.13 | (6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 | via 6,7-dehydrohyoscyamine or not, two possible pathways for the epoxidation in the biosynthesis of scopolamine, overview | Atropa belladonna | scopolamine + succinate + CO2 | - |
? | |
| 1.14.20.13 | 6,7-dehydrohyoscyamine + 2-oxoglutarate + O2 | - |
Atropa belladonna | ? | - |
? | |
| 1.14.20.13 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate + 2-oxoglutarate + O2 | - |
Atropa belladonna | ? | - |
? | |
| 1.14.20.13 | 8-methyl-8-azabicyclo[3.2.1]octan-3-yl benzoate + 2-oxoglutarate + O2 | - |
Atropa belladonna | ? | - |
? | |
| 1.14.20.13 | additional information | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps, the enzyme also catalyzes the reaaction of EC 1.14.11.11. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine | Atropa belladonna | ? | - |
? | |
| 1.14.20.13 | additional information | hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. The catalytic efficiency of AbH6H, especially for the second oxidation, is low. The epoxidation step is much slower than the hydroxylation step. Substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate, and 4 are no substrates for the enzyme | Atropa belladonna | ? | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.14.11.11 | monomer | 1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
| 1.14.20.13 | monomer | 1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation | Atropa belladonna |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.14.11.11 | H6H | - |
Atropa belladonna |
| 1.14.11.11 | hyoscyamine 6beta-hydroxylase | - |
Atropa belladonna |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.14.11.11 | 34 | - |
recombinant enzyme | Atropa belladonna |
| 1.14.20.13 | 34 | - |
recombinant enzyme | Atropa belladonna |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.14.11.11 | 7.6 | - |
recombinant enzyme | Atropa belladonna |
| 1.14.20.13 | 7.6 | - |
recombinant enzyme | Atropa belladonna |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.14.11.11 | additional information | the nitrogen atom in the tropane ring of L-hyoscyamine plays an important role in substrate recognition. Proposed mechanism of epoxidation catalyzed by H6H, overview | Atropa belladonna |
| 1.14.20.13 | additional information | the nitrogen atom in the tropane ring of L-hyoscyamine plays an important role in substrate recognition. Proposed mechanism of epoxidation catalyzed by H6H, overview | Atropa belladonna |
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