Cytochrome P450105D1 (CYP105D1) from Streptomyces griseus: heterologous expression, activity, and activation effects of multiple xenobiotics.
The open reading frame of CYP105D1, a soluble cytochrome P450 from Streptomyces griseus, was cloned behind the tac promoter of the bacterial expression vector pSPg1910L and expressed in Escherichia coli. The recombinant protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. CYP105D1 was active, obtaining reducing equivalents from endogenous E. coli ferredoxin and ferredoxin reductase redox partners present in E. coli. In vitro activity studies revealed CYP105D1 to catalyse the NADH- and NADPH-dependent oxidation of the xenobiotic substrates benzo[a]pyrene, erythromycin, warfarin, and testosterone. Furthermore, this activity could be stimulated in the presence of either alpha-benzoflavone or beta-benzoflavone in an analogous manner to that reported for mammalian P450 forms including human liver cytochrome P4503A4 (CYP3A4). The system produces an alternative to whole-cell biotransformation of xenobiotic for the production of drug metabolites and an experimental system for probing the structural features of a cytochrome P450 with a broad substrate range.
Taylor
M
M
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, SY23 3DA, United Kingdom.
Lamb
D C
DC
Cannell
R
R
Dawson
M
M
Kelly
S L
SL
D000818:Animals; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000201:enzymology; D008322:Mammals; D006899:Mixed Function Oxygenases / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism* / Q000494:pharmacology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C122191:CYP105D1 protein, Streptomyces griseus; D042926:Cytochrome P-450 CYP4A