Comparative Study of Topogenesis of Cytochrome P450scc (CYP11A1) and Its Hybrids with Adrenodoxin Expressed in Escherichia coli Cells

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A. A. Vinogradova, V. N. Luzikov, and L. A. Novikova^*

Belozersky 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 August 28, 2006; Revision received October 6, 2006

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Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH₂- or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH₂-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.

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KEY WORDS: cytochrome P450scc, Escherichia coli, folding, hybrid proteins

DOI: 10.1134/S0006297907020113

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