Steroid 21-hydroxylase, also called steroid 21-monooxygenase, 21α-hydroxylase, P45021A2, and, less commonly 21β-hydroxylase, is a cytochrome P450enzyme that is involved with the biosynthesis of the steroid hormonesaldosterone and cortisol.[1] These syntheses take place in the adrenal cortex.[2] Specifically, 21-hydroxylase converts progesterone and 17α-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively, by hydroxylating at the C21 position.[3] 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.[4] 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.[3] The enzyme is notable for its substrate specificity and relatively high catalytic efficiency.
21-hydroxylase is a complex of three independent and identical enzyme subunits. Each subunit in the human enzyme consists of 13 α-helices and 9 ß-strands, formed into a triangular prism-like tertiary structure.[3] The iron(III) heme group that defines the active site resides in the center of each subunit. The human enzyme binds one substrate at a time.[3] In contrast, the well-characterized bovine enzyme can bind two substrates.[5] The human and bovine enzyme share 80% amino acid sequence identity, but are structurally different, particularly in loop regions, and also evident in secondary structure elements.[3]
21-Hydroxylase is a cytochrome P450 enzyme and follows the P450 catalytic cycle.
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.[7]
Earlier studies of the human enzyme expressed in yeast classified 17-hydroxyprogesterone as the best substrate for 21-hydroxylase.[7][8][9] However, recent analysis of the purified human enzyme found a lower KM and greater catalytic efficiency for progesterone over 17-hydroxyprogesterone.[3]
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.[2] C-H bond breaking to create a primary carbon radical is thought to be the rate-limiting step in the hydroxylation.[3]
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, and occurs in approximately 1 in 15000 births globally.[13][14] There are multiple forms of CAH, broken down into classical and nonclassical forms 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.[15] Cortisol and aldosterone deficits are associated with life-threatening salt-loss (hence salt-wasting), as the steroids play roles in regulating sodiumhomeostasis. 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.[16][17]
↑Auchus RJ, Miller WL (2015). "P450 enzymes in steroid processing". Cytochrome P450: Structure, Mechanism, and Biochemistry (Fourth ed.). Springer International Publishing. pp. 851–879. doi:10.1007/978-3-319-12108-6_12.
↑ 7.07.1Auchus RJ, Sampath Kumar A, Andrew Boswell C, Gupta MK, Bruce K, Rath NP, Covey DF (January 2003). "The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21". Archives of Biochemistry and Biophysics. 409 (1): 134–44. doi:10.1016/s0003-9861(02)00491-5. PMID12464252.
↑Lorence MC, Trant JM, Mason JI, Bhasker CR, Fujii-Kuriyama Y, Estabrook RW, Waterman MR (August 1989). "Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21". Archives of Biochemistry and Biophysics. 273 (1): 79–88. doi:10.1016/0003-9861(89)90164-1. PMID2502949.
↑Wu DA, Hu MC, Chung BC (April 1991). "Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae". DNA and Cell Biology. 10 (3): 201–9. doi:10.1089/dna.1991.10.201. PMID1707279.
White PC, Tusie-Luna MT, New MI, Speiser PW (1994). "Mutations in steroid 21-hydroxylase (CYP21)". Human Mutation. 3 (4): 373–8. doi:10.1002/humu.1380030408. PMID8081391.
Helmberg A (August 1993). "Twin genes and endocrine disease: CYP21 and CYP11B genes". Acta Endocrinologica. 129 (2): 97–108. doi:10.1530/acta.0.1290097. PMID8372604.
de-Araujo M, Sanches MR, Suzuki LA, Guerra G, Farah SB, de-Mello MP (January 1996). "Molecular analysis of CYP21 and C4 genes in Brazilian families with the classical form of steroid 21-hydroxylase deficiency". Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Medicas E Biologicas. 29 (1): 1–13. PMID8731325.
Yu CY (1999). "Molecular genetics of the human MHC complement gene cluster". Experimental and Clinical Immunogenetics. 15 (4): 213–30. doi:10.1159/000019075. PMID10072631.
Forest MG, Tardy V, Nicolino M, David M, Morel Y (June 2005). "21-Hydroxylase deficiency: an exemplary model of the contribution of molecular biology in the understanding and management of the disease". Annales d'Endocrinologie. 66 (3): 225–32. doi:10.1016/s0003-4266(05)81754-8. PMID15988383.
