5vbu
Crystal Structure of Human Cytochrome P450 21A2 Hydroxyprogesterone Complex
Structural highlights
DiseaseCP21A_HUMAN Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, salt wasting form;Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, simple virilizing form. The disease is caused by variants affecting the gene represented in this entry. FunctionCP21A_HUMAN A cytochrome P450 monooxygenase that plays a major role in adrenal steroidogenesis. Catalyzes the hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone to respectively form 11-deoxycorticosterone and 11-deoxycortisol, intermediate metabolites in the biosynthetic pathway of mineralocorticoids and glucocorticoids (PubMed:10602386, PubMed:16984992, PubMed:22014889, PubMed:25855791, PubMed:27721825). Mechanistically, uses molecular oxygen inserting one oxygen atom into a substrate, and reducing the second into a water molecule, with two electrons provided by NADPH via cytochrome P450 reductase (CPR; NADPH-ferrihemoprotein reductase) (PubMed:25855791).[1] [2] [3] [4] [5] Publication Abstract from PubMedCytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17alpha-hydroxyprogesterone (17alpha-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17alpha-OH-progesterone). We found that the 17alpha-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (>/= 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme. Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.,Wang C, Pallan PS, Zhang W, Lei L, Yoshimoto FK, Waterman MR, Egli M, Guengerich FP J Biol Chem. 2017 Jun 30;292(26):10767-10778. doi: 10.1074/jbc.M117.792465. Epub , 2017 May 24. PMID:28539365[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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