TAILIEUCHUNG - Báo cáo khoa học: Critical role of the plasma membrane for expression of mammalian mitochondrial side chain cleavage activity in yeast

Engineered yeast cells efficiently convert ergosta-5-eneol to pregnenolone and progesterone provided that endogenous pregnenolone acetylase activity is disrupted and that heterologous sterolD7-reductase, cytochrome P450 side chain cleavage (CYP11A1) and 3b hydroxysteroid dehydrogenase/isomerase (3b-HSD) activities are present. CYP11A1activity requires the expressionof themammalian NADPH-adrenodoxin reductase (Adrp) and adrenodoxin (Adxp) proteins as electron carriers. | Eur. J. Biochem. 270 1502-1514 2003 FEBS 2003 doi Critical role of the plasma membrane for expression of mammalian mitochondrial side chain cleavage activity in yeast Catherine DuDort 1 Barbara Schoepp1 1 Elise Chatelain1 Roberto Spaanoli2 Bruno Dumas3 and Denis Pompon1 1 Laboratoire d lngenierie des Proteines Membranaires CGM du CNRS Gif sur Yvette France 2Lead Discovery Technologies Aventis Pharma Romainville France 3Functional Genomics Aventis Pharma 13 Quai Jules G uesde F-94403 Vitry sur Seine France Engineered yeast cells efficiently convert ergosta-5-eneol to pregnenolone and progesterone provided that endogenous pregnenolone acetylase activity is disrupted and that heterologous sterol A7-reductase cytochrome P450 side chain cleavage CYP11A1 and 3b hydroxysteroid dehydrogenase isomerase 3b-HSD activities are present. CYP11A1 activity requires the expression of the mammalian NADPH-adrenodoxin reductase Adrp and adrenodoxin Adxp proteins as electron carriers. Several parameters modulate this artificial metabolic pathway the effects of steroid products the availability and delivery of the ergosta-5-eneol substrate to cytochrome P450 electron flux and protein localization. CYP11A1 Adxp and Adrp are usually located in contact with inner mitochondrial membranes and are directed to the outside of the mitochondria by the removal of their respective mitochondrial targeting sequences. CYP11A1 then localizes to the plasma membrane but Adrp and Adxp are detected in the endoplasmic reticulum and cytosol as expected. The electron transfer chain that involves several subcellular compartments may control side chain cleavage activity in yeast. Interestingly Tgl1p a potential ester hydrolase was found to enhance steroid productivity probably through both the availability and or the trafficking of the CYP11A1 substrate. Thus the observation that the highest cellular levels of free ergosta-5-eneol are found in the plasma membrane suggests that .

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