Michael Waterman
Faculty Member
Last active: 2/12/2015

Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.

Richardson TH, Hsu MH, Kronbach T, Barnes HJ, Chan G, Waterman MR, Kemper B, Johnson EF
Arch Biochem Biophys. 1993 300 (1): 510-6

PMID: 8380971 · DOI:10.1006/abbi.1993.1069

Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.

MeSH Terms (24)

Amino Acid Sequence Animals Aryl Hydrocarbon Hydroxylases Base Sequence Cell Membrane Chromatography Chromatography, DEAE-Cellulose Cloning, Molecular Cytochrome P-450 Enzyme System DNA Durapatite Escherichia coli Exons Hydroxyapatites Kinetics Molecular Sequence Data Oligodeoxyribonucleotides Plasmids Polymerase Chain Reaction Rabbits Recombinant Fusion Proteins Recombinant Proteins Steroid 16-alpha-Hydroxylase Steroid Hydroxylases

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