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Literature summary for 1.17.1.5 extracted from
- Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri (1996), Biochemistry, 35, 212-223.
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
- |
Eubacterium barkeri |
General Stability
| General Stability | Organism |
|---|---|
| enzyme is most stable at alkaline pH in the presence of glycerol, 20% glycerol and 400 mM KCl stabilize | Eubacterium barkeri |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| 6-Hydroxynicotinate | inhibits effectively | Eubacterium barkeri |  |
| additional information | not inhibited by incubation for 1 h at room temperature with 100 mM KCN | Eubacterium barkeri | |
| Selenophosphate | 7 mM, 30 min, anaerobic conditions, reversible complete inactivation, time-dependent | Eubacterium barkeri | |
| sodium selenide | 7 mM, 10 min, anaerobic conditions, reversible complete inactivation, time-dependent | Eubacterium barkeri | |
| Sulfide | 1 mM, 10 min, reversible time-dependent inactivation | Eubacterium barkeri | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 23000 | - |
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE | Eubacterium barkeri |
| 33000 | - |
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE | Eubacterium barkeri |
| 37000 | - |
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE | Eubacterium barkeri |
| 50000 | - |
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE | Eubacterium barkeri |
| 160000 | - |
major form, occurence of additional enzyme forms of 400 kDa and 120 kDa with same subunit composition, gel filtration and native PAGE | Eubacterium barkeri |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Eubacterium barkeri | - |
- |
- |
Oxidation Stability
| Oxidation Stability | Organism |
|---|---|
| exposure of substrate-reduced enzyme to air results in a complete loss of activity, enzyme before reduction is much less sensitive to oxygen inactivation | Eubacterium barkeri |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 112fold purification | Eubacterium barkeri |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 18 | - |
- |
Eubacterium barkeri |
Storage Stability
| Storage Stability | Organism |
|---|---|
| room temperature, 50 mM Tris-HCl buffer, pH 8.2, after 1 day 40% loss of hydroxylase activity, after 7 days 62% loss of hydroxylase activity, NADPH oxidase and diaphorase activity of enzyme are more stable | Eubacterium barkeri |
| room temperature, pH8, 9 days, 75% loss of hydroxylase activity | Eubacterium barkeri |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2,3-pyrazinedicarboxylate + H2O + NADP+ | 10-20% of the activity with nicotinate | Eubacterium barkeri | ? | - |
? | |
| 2-pyrazinecarboxylate + H2O + NADP+ | equally as effective as nicotinate | Eubacterium barkeri | ? | - |
? | |
| 3,5-pyridinedicarboxylate + H2O + NADP+ | 5-10% of the activity with nicotinate | Eubacterium barkeri | ? | - |
? | |
| 6-methylnicotinate + H2O + NADP+ | 5-10% of the activity with nicotinate | Eubacterium barkeri | ? | - |
? | |
| nicotinate + H2O + NADP+ | high substrate specificity toward electron donor substrates, unsubstituted nitrogen and a carboxyl group at position 3 are absolutely required for substrate hydroxylation and unsubstituted carbon-5 is important for oxidation at carbon-6 of substrate | Eubacterium barkeri | 6-hydroxynicotinate + NADPH | - |
? | |
| trigonelline + H2O + NADP+ | 5-10% of the activity with nicotinate | Eubacterium barkeri | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| heterotetramer | 1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE | Eubacterium barkeri |
| heterotetramer | 23 kDa protein is less stained and may be a degradation product | Eubacterium barkeri |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| additional information | - |
assay at room temperature | Eubacterium barkeri |
pH Stability
| pH Stability | pH Stability Maximum | Comment | Organism |
|---|---|---|---|
| additional information | - |
maximal stability during incubation for 14 h at room temperature, after 9 days storage at pH 8, 25% of the initial activity retained | Eubacterium barkeri |
| 8 | - |
most stable at alkaline pH | Eubacterium barkeri |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| 4Fe-4S-center | two [2Fe-2S] clusters, 5-7 atoms Fe per 160 kDa enzyme molecule | Eubacterium barkeri | |
| FAD | 1 FAD molecule per 160 kDa protein protomer | Eubacterium barkeri | |
| molybdenum cofactor | - |
Eubacterium barkeri | |
| molybdopterin | Mo is bound to a dinucleotide form of molybdopterin and is coordinated with selenium, 1 mol Mo per 160 kDa enzyme molecule, molybdenum is directly coordinated to selenium, Se-Mo center is required for enzymic oxidation of nicotinate | Eubacterium barkeri | |
| additional information | NAD+ can not replace NADP+, but NADH can replace NADPH | Eubacterium barkeri | |
| additional information | enzyme contains labile selenium cofactor which is essential for hydroxylase activity of enzyme, Se is directly coordinated to Mo, up to 1 Se atom per enzyme molecule | Eubacterium barkeri | |
| NADP+ | - |
Eubacterium barkeri | |
| NADPH | is able to reduce FAD cofactor, NADH can replace NADPH | Eubacterium barkeri | |
| NADPH | NADPH oxidase activity | Eubacterium barkeri | |
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