Expression of Cytochrome P450scc in Escherichia coli Cells: Intracellular Location and Interaction with Bacterial Redox Proteins

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V. M. Shkumatov¹, V. G. Radyuk¹, Y. V. Falertov¹, A. A. Vinogradova², V. N. Luzikov², and L. A. Novikova^2*

¹Institute of Physico-Chemical Problems, State University, Minsk, Belarus

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: novik@genebee.msu.su

^* To whom correspondence should be addressed.

Received March 21, 2006; Revision received April 28, 2006

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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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KEY WORDS: cytochrome P450scc, Escherichia coli, membrane insertion, functional coupling

DOI: 10.1134/S0006297906080104

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