Cloned (Comment) | Organism |
---|---|
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Novosphingobium aromaticivorans |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His-tagged wild-type and mutant enzymes, hanging drop vapour diffusion method, mixing of 0.001 ml of 50 mg/ml protein in crystallisation buffer containing 20 mM Tris, pH 8.0, and 150 mM KCl, with 0.001 ml of reservoir solution containing 0.1 M Tris, pH 8.3, 5% v/v PEG 400, and 1.9 M ammonium sulfate, equilivration against 0.1 ml of reservoir solution, 18°C, X-ray diffraction structure determination and analysis at 2.0 A resolution | Novosphingobium aromaticivorans |
Protein Variants | Comment | Organism |
---|---|---|
L253V | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate | Novosphingobium aromaticivorans |
M98F | site-directed mutagensis, the mutant shows 85% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate | Novosphingobium aromaticivorans |
additional information | substitutions at Tyr96, Met98 and Leu253 in CYP101D2 analogously to closely related CYP101A1 from Pseudomonas putida, reduce both the spin state shift on camphor binding and the camphor oxidation activity | Novosphingobium aromaticivorans |
Y96A | site-directed mutagensis, the mutant shows increased affinity for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane, the monooxygenase activity of the mutant towards alkane substrates is enhanced compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate | Novosphingobium aromaticivorans |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | heme and cytochrome P450 containing enzyme | Novosphingobium aromaticivorans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(+)-camphor + reduced ferredoxin + O2 | Novosphingobium aromaticivorans | - |
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Novosphingobium aromaticivorans | Q2G8A2 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N--terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) | Novosphingobium aromaticivorans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(+)-camphor + reduced ferredoxin + O2 | - |
Novosphingobium aromaticivorans | (+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O | - |
? | |
(+)-camphor + reduced ferredoxin + O2 | - |
Novosphingobium aromaticivorans | (+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O | 99% 5-exo-hydroxycamphor by wild-type enzyme and 92% by Y96A mutant | ? | |
3 2-methylpentane + 3 reduced ferreredoxin + O2 | - |
Novosphingobium aromaticivorans | 2-methyl-pentan-2-ol + 2-methyl-pentan-3-ol + 2-methyl-pentan-4-ol + 3 oxidized ferredoxin | 52.5% 2-methyl-pentan-2-ol + 13% 2-methyl-pentan-3-ol, and 3% 2-methyl-pentan-4-ol for the wild-type enzyme, 5% + 12% + 30% for the mutant Y96A | ? | |
adamantane + reduced ferreredoxin + O2 | - |
Novosphingobium aromaticivorans | 1-adamantol + 2-adamantol + oxidized ferredoxin + H2O | 98% 1-adamantol + 2% 2-adamantol for the wild-type enzyme, 97% + 3% for the mutant Y96A | ? | |
cyclooctane + reduced ferreredoxin + O2 | - |
Novosphingobium aromaticivorans | cyclooctanol + cyclooctanone + oxidized ferredoxin + H2O | 99% cyclooctanol + 1% cyclooctanone for the wild-type enzyme, 97% + 3% for the mutant Y96A | ? | |
hexane + reduced ferreredoxin + O2 | - |
Novosphingobium aromaticivorans | hexan-2-ol + hexan-3-ol + oxidized ferredoxin + H2O | 56% hexan-2-ol + 44% hexan-3-ol for the wild-type enzyme and mutant Y96A | ? | |
additional information | substrate binding and activity data for wild-type CYP101D2 and variants with different substrates, gas chromatography analysis of products, overview | Novosphingobium aromaticivorans | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
CYP101D2 | - |
Novosphingobium aromaticivorans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Novosphingobium aromaticivorans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Novosphingobium aromaticivorans |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
cytochrome P450 | CYP101D2 is a cytochrome P450 monooxygenase | Novosphingobium aromaticivorans | |
Ferredoxin | [2Fe-2S] ferredoxin | Novosphingobium aromaticivorans | |
heme | heme-dependent monooxygenase | Novosphingobium aromaticivorans |
General Information | Comment | Organism |
---|---|---|
additional information | role of Tyr96 in substrate binding | Novosphingobium aromaticivorans |