Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Priestia megaterium |
Crystallization (Comment) | Organism |
---|---|
sitting-drop technique, 4°C, A264Q crystals are obtained with a mother liquor of 100 mM cacodylic acid (pH 6.0), 160 mM MgCl2 and 16% PEG 3350. A264M crystals are obtained with mother liquor of 100 mM cacodylic acid (pH 6.0), 140 mM MgCl2 and 18% PEG 3350. A264C crystals are obtained under the same conditions, but with 100 mM MgCl2. Crystals are flash-frozen in liquid nitrogen using 10% PEG 200 as cryoprotectant. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants | Priestia megaterium |
Protein Variants | Comment | Organism |
---|---|---|
A264C | no notable decreases in expression of the respective haem domains or flavocytochromes. For A264C haem domain, fatty-acid-induced perturbation of the spinstate equilibrium is observed in some cases, but the extent of high-spin conversion is substantially less than observed for wild-type with arachidonate | Priestia megaterium |
A264M | higher dodecanoate affinity than wild-type BM3. For the A264M mutant, a more substantial fatty-acid-induced high-spin conversion of haem iron is observed than for A264C, albeit slightly less than for wild-type with the same fatty acids. | Priestia megaterium |
A264Q | no notable decreases in expression of the respective haem domains or flavocytochromes | Priestia megaterium |
additional information | mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron | Priestia megaterium |
additional information | the position of the amino acid 264 side chain (even in enzymes or subpopulations thereof where the residue does not ligate the haem iron) probably interferes with catalytically productive substrate docking | Priestia megaterium |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
4-phenylimidazole | inhibitory azole ligand that co-ordinates the haem iron in the sixth (axial) position | Priestia megaterium |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Iron | investigation of the haem-domain | Priestia megaterium |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Priestia megaterium | P14779 | expression in Escherichia coli | - |
Purification (Comment) | Organism |
---|---|
homogeneity, chromatographic methods (DEAE-Sepharose, Q-Sepharose and hydroxyapatite resins) | Priestia megaterium |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | fatty-acid-stimulated electron transfer in wild-type and Ala264 mutant flavocytochromes is linked to substrate hydroxylation | Priestia megaterium | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPHcytochrome P450 reductase and P450 domains | Priestia megaterium |
Synonyms | Comment | Organism |
---|---|---|
flavocytochrome P450 BM3 | - |
Priestia megaterium |
P450 BM3 | bifunctional P-450/NADPH-P450 reductase, EC1.14.14.1/EC1.6.2.4 | Priestia megaterium |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | - |
Priestia megaterium |