Structure and expression of the CYP21 (P450c21, steroid 21-hydroxylase) gene with respect to its deficiency

Abstract

Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.

Function and membrane topology of wild-type and mutated cytochrome P-450c21

“…We studied the membrane topology and function of P450c21 with a Pro-30 to Leu substitution. The Pro-30 to Leu mutation was identified in patients suffering from the non-classical type of P450c21 deficiency [29,39]. The lower activity of L30 is consistent with the phenotype observed in the non-classical type of the disease due to P450c21 deficiency.…”

The biosynthesis of steroids and triterpenoids

“…[813][814][815][816][817][818][819][820][821] The clinical condition congenital adrenal hyperplasia is over 90% associated with deficiency of this enzyme 822,823 and a rapid PCR protocol for determination of mutations in 21-hydroxylase has been described. 824,825 From mutation studies, Cys-428 has been proposed to be the heme ligand and the presence of a methyl group at the -carbon of Val-281 has been shown to be required for heme-incorporation and consequent enzymatic activity. 826 Mutations in Ile-172 (believed to be involved in the membrane-anchoring domain) and Arg-356 in the substratebinding site resulted in loss of activity.…”

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

“…Because of their proximity, these two genes exchange their sequences frequently through gene conversion events. This gene conversion is the main cause for the mutations found in the c21B gene (16) and can be used as the basis for the detection of known mutations in patients (17).…”