Purification and Characterization of Recombinant-Expressed Cytochrome P450 2C3 from Escherichia coli: 2C3 Encodes the 6β-Hydroxylase Deficient form of P450 3b

T. H. Richardson, M. H. Hsu, T. Kronbach, H. J. Barnes, G. Chan, M. R. Waterman, B. Kemper, E. F. Johnson

Research output: Contribution to journalArticlepeer-review

Abstract

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 17α. The nucleotide sequence encoding the N-terminus of P450 17α was modified previously to achieve a high level of expression of P450 17α in E. coli by altering the first eight codons of P450 17α 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-α-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16α, but not 6β-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6β-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5β-pregnane-3β,20α-diol, with a resultant V(max) = 10 min-1 and K(m) = 20 μM and is not inhibited by 16α-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 16α- and 6β-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.

Original languageEnglish (US)
Pages (from-to)510-516
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume300
Issue number1
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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