Expression and Functional Study of Wild-Type and Mutant Human Cytochrome P450c21 inSaccharomyces cerevisiae

Abstract

The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.

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

“…pombe, with steroid hydroxylation rates being significantly higher for the conversion of 17α-hydroxyprogesterone to 11-deoxycortisol compared to that of progesterone to 11-deoxycorticosterone (Figure 1). This finding correlates well with former reports, where the apparent v max for 17α-hydroxyprogesterone was also considerably higher than for progesterone (Lorence et al, 1989;Wu et al, 1991). Unexpectedly, CYP21-expressing cells showed lower oxygen consumption than the parental strain, even in the presence of the steroid substrates (Figure 2).…”

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

“…The mutated cDNA was subcloned into p5Јc21 and pYE8 vectors for in vitro transcription/translation and yeast expression, respectively, as described before (7,25).…”

Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively.

“…The calcium-phosphate precipitation method was used to transfect COS-1 cells with 21-hydroxylase cDNA (phc2 1) and a ,-galactosidase gene (RSV-#-gal) which was used as an internal control to normalize the transfection efficiency (21). 2 d after transfection, cells were incubated with 1 AsM ['4C]-17-hydroxyprogesterone for 1-24 h. Steroids in the media were extracted and the cells were harvested for protein immunoblotting assays (21).…”

“…pYE8 vector contains the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the 2 origin of replication (22). Transformation of plasmids into S. cerevisiae strain 20B-12 has been described previously (21,23).…”