Literature summary for 1.6.2.4 extracted from

Girvan, H.M.; Toogood, H.; Littleford, R.E.; Seward, H.E.; Smith, W.E.; Ekanem, I.; Leys, D.; Cheesman, M.; Munro, A.W.
Novel heme coordination variants of flavocytochrome P450 BM3 (2009), Biochem. J., 417, 65-76.

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Priestia megaterium

Crystallization (Commentary)

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

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

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

Inhibitors	Comment	Organism	Structure
4-phenylimidazole	inhibitory azole ligand that co-ordinates the haem iron in the sixth (axial) position	Priestia megaterium

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Iron	investigation of the haem-domain	Priestia megaterium

Organism

Organism	UniProt	Comment	Textmining
Priestia megaterium	P14779	expression in Escherichia coli	-

Purification (Commentary)

Purification (Comment)	Organism
homogeneity, chromatographic methods (DEAE-Sepharose, Q-Sepharose and hydroxyapatite resins)	Priestia megaterium

Substrates and Products (Substrate)

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

Subunits	Comment	Organism
More	self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPHcytochrome P450 reductase and P450 domains	Priestia megaterium

Synonyms

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

Cofactor	Comment	Organism	Structure
FMN	-	Priestia megaterium