Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.
The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.
Green
A J
AJ
Department of Pure & Applied Chemistry, University of Strathclyde, The Royal College, Glasgow, UK.
Rivers
S L
SL
Cheeseman
M
M
Reid
G A
GA
Quaroni
L G
LG
Macdonald
I D
ID
Chapman
S K
SK
Munro
A W
AW
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin / Q000096:biosynthesis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
C445101:12-imidazolyldodecanoic acid; D001426:Bacterial Proteins; C428043:BioI protein, Bacillus subtilis; D005227:Fatty Acids; D007093:Imidazoles; C445100:para-nitrophenoxydodecanoic acid; D019814:Myristic Acid; D001710:Biotin; C037738:1-phenylimidazole; D003577:Cytochrome P-450 Enzyme System; C117025:pentadecanoic acid