Expression of Bovine Cytochrome P450c21 and Its Fused Enzymes with Yeast NADPH-Cytochrome P450 Reductase inSaccharomyces cerevisiae

Abstract

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

“…The Ser-Ser-Thr linker used in this construct was chosen to enable cloning and has been previously described 43 in a study on the influence of different linker groups on the enzymatic activity of fusion proteins. The conclusion reached was that the number or the charge of amino acids in the linker region had a small effect, up to two-fold, on the rate of catalysis of the fusion protein.…”

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

“…The maximal conversion rate of progesterone is attained during the first 24 h ( Figure 1C) and was determined to be 21 ± 1 µM/day [or 1.26 ± 0.06 µmol/g (CWW)/day], while for 17α-hydroxyprogesterone the rate was 259 ± 9 µM/day [or 15.6 ± 0.5 µmol/g (CWW)/d], which is one order of magnitude higher than for the former substrate. Biotransformations with recombinant baker's yeast expressing bovine CYP21 reported previously (Sakaki et al, 1990(Sakaki et al, , 1991Szczebara et al, 2003) might have had significantly lower steroid conversion rates, but the experimental setup was not entirely comparable. Nevertheless, the strong steroid hydroxylation activity of fission yeast strain CAD18 demonstrates its usefulness as a eukaryotic model for P450-driven biocatalysis.…”

Expression of rat liver vitamin D3 25‐hydroxylase cDNA in Saccharomyces cerevisiae

“…However, functional expression of a mitochondrial P450 species in the yeast has not succeed- [11] was used for construction of an expresston plasmid for P450LMT2S, A P450 hemoprotein in transformed yeast cells was measured by reduced CO-difference spectra [10], Yeast cellular proteins were analyzed by Western immunoblotting using anti-rat P450LMT~5 IG and [tZSl]anti-mouse IG F(ab')z fragment (Amersham Japan, Tokyo) as described [7], Yeast spheroplasts were prepared [101, and then subjected to subcellular fractionation [12]. P450LMrzs-dependent monooxygenase activities toward Ia.…”