Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae.
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.
Wu
D A
DA
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Hu
M C
MC
Chung
B C
BC
D015152:Blotting, Northern; D015153:Blotting, Western; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009154:Mutation; D010957:Plasmids; D012313:RNA / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013057:Spectrum Analysis; D013255:Steroid 21-Hydroxylase / Q000737:chemistry / Q000235:genetics*
D012313:RNA; D013255:Steroid 21-Hydroxylase