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Literature summary for 5.4.99.1 extracted from
- Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway (2015), Metab. Eng., 30, 190-196 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| genes mutS and mutE, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) pLysS | Clostridium tetanomorphum |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | production of mesaconate in Escherichia coli by engineered glutamate mutase pathway, establishment of mesaconate pathway in Escherichia coli. First, glutamate is synthesized from glucose via glycolysis and TCA cycle.Then glutamate is converted into 3-methylaspartate by glutamate mutase. Finally, mesaconate is formed by elimination of ammonia from 3-methylaspartate via MAL. Since Escherichia coli does not contain glutamate mutase and 3-methylaspartate ammonia lyase, the two enzymes from Clostridium tetanomorphum are heterologously expressed. To increase the flux from glutamate to mesaconate, two effective strategies are employed to optimize the critical enzyme activity in the pathway: one is regenerating inactive mutase. The other is enhancing the availability of glutamate mutase (stability) and coenzyme B12 (regeneration). For the highest mesaconate production strain EM9, the consumed glutamate is 6.91 g/l (40.9 mM) and mesaconate titer is 7.81 g/l (60 mM). The GlmE from Clostridium cochlearium shows best performance in mesaconate titer because GlmE is more stable than MutE | Clostridium tetanomorphum |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| L-threo-3-methylaspartate | Clostridium tetanomorphum | - |
L-glutamate | - |
r |  |
| L-threo-3-methylaspartate | Clostridium tetanomorphum ATCC 15920 | - |
L-glutamate | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Clostridium tetanomorphum | Q05488 AND Q05509 | small sigma subunit and large epsilon subunit, respectively | - |
| Clostridium tetanomorphum ATCC 15920 | Q05488 AND Q05509 | small sigma subunit and large epsilon subunit, respectively | - |
| no activity in Escherichia coli | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme from Escherichia coli strain Rosetta2 (DE3) pLysS by nickel affinity chromatograpyh and ultrafiltration, recombinant expression in Escherichia coli strain BW25113, coexpression with Clostridium tetanomorphum 3-methylaspartate ammonia lyase | Clostridium tetanomorphum |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| L-threo-3-methylaspartate | - |
Clostridium tetanomorphum | L-glutamate | - |
r | |
| L-threo-3-methylaspartate | - |
Clostridium tetanomorphum ATCC 15920 | L-glutamate | - |
r | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| heterodimer | the enzyme contains a small sigma subunit and a large epsilon subunit | Clostridium tetanomorphum |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Glutamate mutase | - |
Clostridium tetanomorphum |
| mutE | gene name, large subunit | Clostridium tetanomorphum |
| mutS | gene name, small subunit | Clostridium tetanomorphum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Clostridium tetanomorphum |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Clostridium tetanomorphum |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| coenzyme B12 | adenosylcobalamin, the concentration of internal coenzyme B12 is crucial for the full activity of B12-dependent enzyme | Clostridium tetanomorphum | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | identification of the reactivating factor of glutamate mutase, MutL. MutL regenerates glutamate mutase and releases inactive coenzyme from the inactive glutamate mutase-adenosylcobalamine complex. Reactivation of the spontaneous inactive mutase (mutase-X-Cbl) and inactive form B12 binding with mutase (mutase-CNCbl) in presence of ATP and coenzyme B12 by MutL | Clostridium tetanomorphum |
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