Expression of cytochrome P450scc in Escherichia coli cells: intracellular location and interaction with bacterial redox proteins.
Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.
Shkumatov
V M
VM
Institute of Physico-Chemical Problems, State University, Minsk, Belarus.
Radyuk
V G
VG
Falertov
Y V
YV
Vinogradova
A A
AA
Luzikov
V N
VN
Novikova
L A
LA
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002786:Cholesterol Side-Chain Cleavage Enzyme