Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017alpha.
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.
Shkumatov
V M
VM
Research Institute for Physical Chemical Problems, Belarussian State University, Minsk, 220050, Belarus. biopharm@bsu.by
Usova
E V
EV
Poljakov
Y S
YS
Frolova
N S
NS
Radyuk
V G
VG
Mauersberger
S
S
Chernogolov
A A
AA
Honeck
H
H
Schunck
W-H
WH
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D001711:Biotransformation; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006908:Hydroxyprogesterones / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis / Q000378:metabolism*
D006908:Hydroxyprogesterones; D013256:Steroids; D011374:Progesterone; D013254:Steroid 17-alpha-Hydroxylase