Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017 α

Tools

Item Type:	Article
Title:	Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017 α
Creators Name:	Shkumatov, V.M., Usova, E.V., Poljakov, Y.S., Frolova, N.S., Radyuk, V.G., Mauersberger, S., Chernogolov, A.A., Honeck, H. and Schunck, W.H.
Abstract:	The cDNA encoding cytochrome P-45017{alpha} from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017{alpha} per liter of the culture. Binding of progesterone to the cytochrome P-45017{alpha} was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17{alpha}-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017{alpha} and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17{alpha}-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017{alpha}-dependent hydroxylation reaction. The reduction of the 20-keto group of 17{alpha}-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017{alpha} expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11{beta}- > 11{alpha}- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017{alpha} and further application of enzymatic properties of this protein to perform specific steroid biotransformations.
Keywords:	Cytochrome P-45017{alpha}, Recombinant Yeast S. Cerevisiae, 17{alpha}-Hydroxyprogesterone, 17{alpha}-Hydroxy-20-Dihydroprogesteron E, 20-Ketosteroid Reductase, Animals, Cattle
Source:	Biochemistry Moskau
ISSN:	0006-2979
Publisher:	MAIK Nauka/Interperiodica
Volume:	67
Number:	4
Page Range:	456-467
Date:	April 2002
Official Publication:	https://doi.org/10.1023/A:1015290108071
PubMed:	View item in PubMed

Repository Staff Only: item control page