Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins |
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Authors: | V. M. Shkumatov V. G. Radyuk Y. V. Falertov A. A. Vinogradova V. N. Luzikov L. A. Novikova |
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Affiliation: | (1) Institute of Physico-Chemical Problems, State University, Minsk, Belarus;(2) Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia |
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Abstract: | 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. |
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Keywords: | cytochrome P450scc Escherichia coli membrane insertion functional coupling |
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