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Literature summary for 1.3.99.31 extracted from
- Structural and kinetics properties of a mutated phytoene desaturase from Rubrivivax gelatinosus with modified product specificity (2011), Arch. Biochem. Biophys., 505, 118-122.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression in Escherichia coli | Rubrivivax gelatinosus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| L153P/L278P | increased formation of neurosporene compared to wild-type enzyme. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg), the ratio of the mutant enzyme is 12:88 | Rubrivivax gelatinosus |
| L208F | increased formation of lycopene compared to wild-type enzyme. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 26:74 after complementation of CrtI in pPEU(DH5a/pACCrtEBEU/pPEUCrtIRg), the ratio of the mutant enzyme is 89:11 | Rubrivivax gelatinosus |
| L208P | increased formation of neurosporene compared to wild-type enzyme. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg), the ratio of the mutant enzyme is 3:97 | Rubrivivax gelatinosus |
| additional information | two different mutation libraries for the crtI gene are constructed to screen for modified enzymes which synthesize almost exclusively either neurosporene or lycopene. The resulting mutants carry between one and four amino acid exchanges and at least one of them affects the secondary protein structure by shortening or extending one of the helices. A prominent amino acid which is exchanged in the neurosporene or lycopene-forming desaturase is leucine 208. Enzyme kinetic studies are carried out with the L208 modified desaturase and the specificities for phytoene and neurosporene as substrates determined. Higher and lower values correlate well with the higher or lower potential for the synthesis of lycopene from neurosporene. TopPred analysis of the mutations of L208 indicate that the location is in a highly hydrophobic membrane-integrated region which is a good candidate for the substrate-binding site of the desaturase | Rubrivivax gelatinosus |
| T256M/D355GL424P | increased formation of neurosporene compared to wild-type enzyme. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg), the ratio of the mutant enzyme is 0:100 | Rubrivivax gelatinosus |
| Y44C/D53G/P134L/V395A | increased formation of neurosporene compared to wild-type enzyme. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg), the ratio of the mutant enzyme is 3:97 | Rubrivivax gelatinosus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 4.8 | - |
15-cis-phytoene | pH 8, 28°C, mutant enzyme L208F (complementation of CrtI in pPEU (DH5a/pACCrtEBEU/pPEUCrtIRg)) | Rubrivivax gelatinosus |  |
| 14.8 | - |
15-cis-phytoene | pH 8, 28°C, wild-type enzyme | Rubrivivax gelatinosus | |
| 17.1 | - |
15-cis-phytoene | pH 8, 28°C, mutant enzyme L208P (complementation of CrtI in pUC8 (DH5a/pACCrtEBEU/pUCCrtIRg)) | Rubrivivax gelatinosus | |
| 33.1 | - |
all-trans-neurosporene | pH 8, 28°C, wild-type enzyme | Rubrivivax gelatinosus | |
| 41.7 | - |
all-trans-neurosporene | pH 8, 28°C, mutant enzyme (complementation of CrtI in pUC8 (DH5a/pACCrtEBEU/pUCCrtIRg)) | Rubrivivax gelatinosus | |
| 65.8 | - |
all-trans-neurosporene | pH 8, 28°C, mutant enzyme L208F (complementation of CrtI in pPEU (DH5a/pACCrtEBEU/pPEUCrtIRg)) | Rubrivivax gelatinosus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 15-cis-phytoene + 3 acceptor | Rubrivivax gelatinosus | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively | all-trans-neurosporene + 3 reduced acceptor | - |
? | |
| 15-cis-phytoene + 4 acceptor | Rubrivivax gelatinosus | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively | all-trans-lycopene + 4 reduced acceptor | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Rubrivivax gelatinosus | - |
- |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 15-cis-phytoene + 3 acceptor | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively | Rubrivivax gelatinosus | all-trans-neurosporene + 3 reduced acceptor | - |
? | |
| 15-cis-phytoene + 3 acceptor | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg) and 26:74 after complementation of CrtI in pPEU(DH5a/pACCrtEBEU/pPEUCrtIRg). The affinity for neurosporene conversion is poorer than for phytoene conversion. This explains the formation of two desaturation products | Rubrivivax gelatinosus | all-trans-neurosporene + 3 reduced acceptor | - |
? | |
| 15-cis-phytoene + 4 acceptor | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively | Rubrivivax gelatinosus | all-trans-lycopene + 4 reduced acceptor | - |
? | |
| 15-cis-phytoene + 4 acceptor | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. The ratio of lycopene to neurosporene of the wild-type enzyme under assay conditions is 88:12 after complementation of CrtI in pUC8(DH5a/pACCrtEBEU/pUCCrtIRg) and 26:74 after complementation of CrtI in pPEU(DH5a/pACCrtEBEU/pPEUCrtIRg). The affinity for neurosporene conversion is poorer than for phytoene conversion. This explains the formation of two desaturation products | Rubrivivax gelatinosus | all-trans-lycopene + 4 reduced acceptor | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CrtI | - |
Rubrivivax gelatinosus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 28 | - |
assay at | Rubrivivax gelatinosus |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Rubrivivax gelatinosus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | added to the reaction mixture | Rubrivivax gelatinosus | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | the enzyme simultaneously catalyzes a three- and four-step desaturation of phytoene producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively | Rubrivivax gelatinosus |
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Release 2023.1 (January 2023)
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