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Steroid 21-hydroxylase is a cytochrome P450 enzyme integral to the biosynthesis of the steroid hormones aldosterone and cortisol. These syntheses take place in the adrenal cortex. Specifically, 21-hydroxylase converts progesterone and 17α-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively, by hydroxylating at the C21 position. The products of the conversions then continue through their appropriate pathways towards creation of aldosterone and cortisol. Like other cytochrome P450 enzymes, 21-hydroxylase participates in the cytochrome P450 catalytic cycle, and engages in one-electron transfer with NADPH-P450 reductase. Its structure includes an essential iron heme group centered within the protein, also common to all P450 enzymes. Variations of the 21-hydroxylase enzyme can be found in all vertebrates. However, understanding of human 21-hydroxylase structure and function is of particular clinical value, as a failure of the enzyme to act appropriately results in congenital adrenal hyperplasia. The x-ray crystal structure for human 21-hydroxylase, with bound progesterone, was realized and published in 2015, providing opportunity for further study. The enzyme is notable for its substrate specificity and relatively high catalytic efficiency.

Function
21-hydroxylase is a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. The 21-hydroxylase enzyme is one of three microsomal steroidogenic P450 enzymes, the others being 17-hydroxylase and aromatase. This protein localizes to the endoplasmic reticulum and hydroxylates steroids at the C21 position. Its activity is required for the synthesis of cortisol and aldosterone.

Kinetics
21-hydroxylase is highly specific for hydroxylation of progesterone and 17-hydroxyprogesterone. No studies have reported sufficient binding of alternate substrates. In this way, it differs from the evolutionarily and functionally related P450 enzyme 17-hydroxylase, which has a large range of substrates.

Earlier studies of the human enzyme expressed in yeast classified 17-hydroxyprogesterone as the best substrate for 21-hydroxylase. However, recent analysis of the purified human enzyme found a lower KM and greater catalytic efficiency for progesterone over 17-hydroxyprogesterone.

The 2015 analysis found the catalytic efficiency of 21-hydroxylase for conversion of progesterone in humans to be approximately 1.3 x 10^7 M-1s-1 at 37 °C. This makes it the most catalytically efficient P450 enzyme of those reported, as of 2015, and more catalytically efficient than the closely related bovine 21-hydroxylase enzyme. C-H bond breaking to create a primary carbon radical is thought to be the rate-limiting step in the hydroxylation.

Clinical significance
In humans, 21-Hydroxylase is encoded by the gene CYP21A2. A related pseudogene is located near this gene; gene conversion events involving the functional gene and the pseudogene are thought to account for many cases of steroid 21-hydroxylase deficiency. Both genes are located on chromosome 6, in the major histocompatibility complex, and the pseudogene, CYP21A1, retains 98% exonic sequence identity with the functional gene.

A defect within the CYP21A2 gene causes a disturbance of the development of the enzyme, which leads to congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, and occurs in approximately 1 in 15000 births globally. There are multiple forms of CAH, broken down into classical and nonclassical based on the amount of function retained. The classical forms include salt-wasting (SW), and simple-viralizing (SV). Mutations that interfere with the active site--the heme group or residues involved in substrate binding--result in a complete loss of enzymatic activity, the salt-wasting type. Cortisol and aldosterone deficits are associated with life-threatening salt-loss (hence salt-wasting), as the steroids play roles in regulating sodium homeostasis. Retaining minimal enzyme activity, the simple-viralizing type, is associated with mutations in conserved hydrophobic regions or near the transmembrane domain. Simple viralizing CAH patients maintain adequate sodium homeostasis, but exhibit other phenotypical symptoms shared by SW, including accelerated growth in childhood and ambiguous genitalia in female neonates. Nonclassical forms retain 20-60% of hydroxylase function--this form is associated with normal cortisol expression, but an excess of androgens post-puberty.

Names and classification
21-Hydroxylase is also called steroid 21-monooxygenase, 21α-Hydroxylase, P450 21A2 and, less commonly 21β-Hydroxylase.

Reaction
21-Hydroxylase catalyzes the addition of hydroxyl (-OH) to the C21 position of two steroids: progesterone and 17α-hydroxyprogesterone. The overall reactions catalyzed by 21-hydroxylase are below.