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Abstract

The cDNA encoding cytochrome P-45017alpha 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-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha 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 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha 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 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-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-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

Protein Engineering of the Progesterone Hydroxylating P450‐Monooxygenase CYP17A1 Alters Its Regioselectivity

“…The formation of the second product was described previously by Szczebara et al . As it is not a natural product of the investigated enzyme, they searched analogue enzymes to mammalian 20α‐hydroxysteroid‐dehydrogenase (20α‐HSD) and found two possible candidates in S. cerevisiae : Ypr1p, a yeast aldo‐keto reductase, and Gcy1p, which is also known as yeast galactose‐inducible crystalline‐like protein . Although the second product is formed by a different enzyme, both steroids need to be analysed and considered upon investigating the activity of the CYP17A1 enzyme.…”

“…In this study, the general activity of the bovine CYP17A1 was investigated by performing whole‐cell biocatalysis with Saccharomyces cerevisiae as a host organism and the YEp5117α construct, described by Shkumatov et al . For the wild type, 95.6 % conversion of the substrate was reached after 24 h with 100 μ m progesterone (Figure S1).…”

“…Plasmid : The used plasmid YEp5117α was described before by Shkumatov et al . This plasmid bears the cDNA gene encoding CYP17A1 from the bovine adrenal cortex microsomes.…”

“…Whole‐cell biocatalysis : Whole‐cell biocatalysis was performed according to Shkumatov et al. with slight modifications . For cultivation, yeast extract‐peptone‐dextrose (YPD, 20 mL) medium was inoculated with a single colony picked from freshly transformed S. cerevisiae cells plated on selective plates.…”

“…Spectral determination of P450 in whole yeast cells : For the spectral determination samples of the single variants, double variants as well as wild type and empty control were grown in 50 mL as described above, without the addition of substrate. The cells were harvested at 3000 g for 15 min at 4 °C and were stored at −20 °C until further use.…”