Heterologous expression, purification, and properties of human cytochrome P450 27C1.
Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.
Wu
Zhong-Liu
ZL
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Bartleson
Cheryl J
CJ
Ham
Amy-Joan L
AJ
Guengerich
F Peter
FP
D000325:Adrenodoxin / Q000737:chemistry; D000595:Amino Acid Sequence; D002112:Calcifediol / Q000737:chemistry; D002384:Catalysis; D002762:Cholecalciferol / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000072501:Cytochrome P450 Family 27; D004926:Escherichia coli / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000325:Adrenodoxin; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; C508522:CYP27C1 protein, human; D000072501:Cytochrome P450 Family 27; D005287:Ferredoxin-NADP Reductase; D002112:Calcifediol