Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Plexaura homomalla |
Crystallization (Comment) | Organism |
---|---|
purified enzyme in complex with arachidonic acid, anaerobic conditions, vapor diffusion with a well solution of 8% PEG-8000, 5% glycerol, 0.2 M CaCl2, 0.1 M imidazole acetate, pH 8.0, crystals are soaked for about 17 h in a solution consisting of 25% glycerol, 10% PEG-8000, 0.02 M CaCl2, 0.1 M imidazole acetate, pH 8.0, 1% dimethyl sulfoxide, and 1 mg/ml arachindonic acid, X-ray diffraction structure determination and analysis at 2.0 A resolution | Plexaura homomalla |
Protein Variants | Comment | Organism |
---|---|---|
A589M | site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme | Plexaura homomalla |
A620H | site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme | Plexaura homomalla |
R182A | site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme | Plexaura homomalla |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | substrate inhibition steady state kinetics | Plexaura homomalla | |
0.03 | - |
arachidonate | recombinant His-tagged wild-type enzyme, pH 7.4, 22°C | Plexaura homomalla | |
0.058 | - |
arachidonate | recombinant His-tagged mutant A620H, pH 7.4, 22°C | Plexaura homomalla | |
0.059 | - |
arachidonate | recombinant His-tagged mutant A589M, pH 7.4, 22°C | Plexaura homomalla | |
0.067 | - |
arachidonate | recombinant His-tagged mutant R182A, pH 7.4, 22°C | Plexaura homomalla |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | the catalytic iron in 8R-LOX is positioned by three invariant His384, His389, and His570 side chains and the terminal main chain. Fe2+ sits in the base of a large U-shaped cavity, positioned by invariant Leu385 on one side, and the iron and His384 and His389 on the other. Leu385 and the catalytic iron cradle the base of the U | Plexaura homomalla |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
arachidonate + O2 | Plexaura homomalla | - |
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Plexaura homomalla | O16025 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) | Plexaura homomalla |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
arachidonate + O2 | - |
Plexaura homomalla | (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate | - |
? | |
arachidonate + O2 | subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation | Plexaura homomalla | (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate | - |
? | |
additional information | in the absence of arachidonate, Tyr181 and Arg182 of helix alpha2 participate in an interhelical charge cluster with Glu430 of the arched helix | Plexaura homomalla | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
8R-LOX | - |
Plexaura homomalla |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Plexaura homomalla |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
3.2 | - |
arachidonate | recombinant His-tagged mutant A620H, pH 7.4, 22°C | Plexaura homomalla | |
32 | - |
arachidonate | recombinant His-tagged mutant A589M, pH 7.4, 22°C | Plexaura homomalla | |
193 | - |
arachidonate | recombinant His-tagged mutant R182A, pH 7.4, 22°C | Plexaura homomalla | |
210 | - |
arachidonate | recombinant His-tagged wild-type enzyme, pH 7.4, 22°C | Plexaura homomalla |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | 8 | assay at | Plexaura homomalla |
General Information | Comment | Organism |
---|---|---|
additional information | subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation, both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery, modeling, overview | Plexaura homomalla |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
55.2 | - |
arachidonate | recombinant His-tagged mutant A620H, pH 7.4, 22°C | Plexaura homomalla | |
542.4 | - |
arachidonate | recombinant His-tagged mutant A589M, pH 7.4, 22°C | Plexaura homomalla | |
2880.6 | - |
arachidonate | recombinant His-tagged mutant R182A, pH 7.4, 22°C | Plexaura homomalla | |
7000 | - |
arachidonate | recombinant His-tagged wild-type enzyme, pH 7.4, 22°C | Plexaura homomalla |