The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function.
A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5. The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b5. Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235.
P L
PL
Holmans
M S
MS
Shet
C A
CA
Martin-Wixtrom
C W
CW
Fisher
R W
RW
Estabrook
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011374:Progesterone / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013739:Testosterone / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins; D013739:Testosterone; D011374:Progesterone; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase