Heterologous expression, purification, and properties of human cytochrome P450 27C1.

Wu ZL, Bartleson CJ, Ham AJ, Guengerich FP
Arch Biochem Biophys. 2006 445 (1): 138-46

PMID: 16360114 · DOI:10.1016/j.abb.2005.11.002

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.

MeSH Terms (20)

Adrenodoxin Amino Acid Sequence Calcifediol Catalysis Cholecalciferol Cholesterol Cloning, Molecular Cytochrome P-450 Enzyme System Cytochrome P450 Family 27 Escherichia coli Ferredoxin-NADP Reductase Humans Mass Spectrometry Mitochondria Molecular Sequence Data Mutation Organ Specificity Peptide Fragments Recombinant Proteins RNA, Messenger

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