Abstract
Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.
Highlights
P450 21A2 catalyzes 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone, an important step in adrenal steroidogenesis
Purification of Human P450 21A2 and Catalytic Properties— To facilitate the expression of human P450 21A2 in E. coli, the N terminus was replaced with the sequence MAKKTSSKGK to increase the solubility of this enzyme; this sequence has been utilized to improve heterologous expression of several P450s [27]
A small 420 nm peak was observed in the assays (when 1 M safranin T was added as a mediator to increase the rate of reduction [29]), indicating that the purified human P450 21A2 is very low in the presence of the inactive form (Ͻ5%)
Summary
P450 21A2 catalyzes 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone, an important step in adrenal steroidogenesis. Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ϳ95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. We have obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. Cytochrome P450 (P450)4 21A2 is the major steroid 21-hydroxylase, which catalyzes the 21-hydroxylation of progesterone and 17␣-hydroxyprogesterone (17␣-OH-progesterone) to form 11-deoxycorticosterone and 11-deoxycortisol, respectively (Fig. 1), primarily in the adrenal cortex [1,2,3]. There are two CYP21 genes in the human genome, CYP21A1 (a pseudogene) and CYP21A2 (the functional gene) [8, 9] Both are located on chromosome 6 in the major histocompatibility locus. The worldwide incidence of congenital adrenal hyperplasia is 1:15,000 for the classical form and at least 1:1,000 for the nonclassical form [7, 10], due primarily to gene conversion events with the adjacent CYP21A1P pseudogene; congenital adrenal hyperplasia is one of the most common inborn meta-
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