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Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli

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Abstract

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR–Adx–P450scc to P450scc–Adx–AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05–0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

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Acknowledgements

The authors would like to thank M. S. Serebryakova for conducting the mass spectrometry experiments, and V. N. Tashlitsky for conducting the experiments using the HPLC.

Funding

This research was supported by Russian Foundation for Basic Research (Grant No. 16-54-00139).

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Authors and Affiliations

  1. Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, 119234, Russia

    V. S. Efimova & M. A. Rubtsov

  2. A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/40, Moscow, 119234, Russia

    L. V. Isaeva & L. A. Novikova

  3. Department of Biochemistry, I.M. Sechenov First Moscow State Medical University (Sechenov University), Trubetskaya, 8, Moscow, 119991, Russia

    M. A. Rubtsov

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Efimova, V.S., Isaeva, L.V., Rubtsov, M.A. et al. Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli. Mol Biotechnol 61, 261–273 (2019). https://doi.org/10.1007/s12033-019-00158-6

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