Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Abstract

Edited by Ruma BanerjeeCytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17␣-hydroxyprogesterone (17␣-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 (17␣-OH-progesterone). We found that the 17␣-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 saltwasting 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 (K s ) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b 5 , 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-d 3 -progesterone, indicating that C-H bond breaking is a ratelimiting step over a 10 4 -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.Cytochrome P450 (P450 or CYP) 5 21A2 (1), an enzyme located in the adrenal cortex, catalyzes the 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone (17␣-OHprogesterone), forming 11-deoxycorticosterone and 11-deoxycortisol, respectively (2-4). Deficiencies in the CYP21A2 gene are common and are involved in ϳ95% of the cases of congenital adrenal hyperplasia (CAH) (2, 3, 5-7), one of the most common inborn errors of metabolism. The incidence of CAH is ϳ1:15,000 worldwide, with well over 100 different genetic variants having been diagnosed. The phenotypes are generally classified as nonclassical (NC), simple virilizing (SV), and salt-wasting (SW), in order of increasing severity (2, 3, 6). The great majority of mutations in CYP21A2 can be traced to the nonfunctional pseudogene of P450 21A2 (CYP21A1P), which is found in Ͼ 70% of all Caucasians. The pseudogene is 98% identical to the functional gene, and crossovers, sequence exchanges, and apparent gene conversion between functional gene and pseudogene contribute to frequent variations in the P450 21A2 protein and the occurrence of CAH (2, 3, 6, 7).We published a crystal structure of bovine P450 21A2 bound with the substrate 17␣-OH-progesterone (8), which prov...

Identification and circumvention of bottlenecks in CYP21A2‐mediated premedrol production using recombinant Escherichia coli

“…By contrast, a previous study described an inhibitory effect of cytochrome b 5 on the CYP21A2 activity in vitro (Wang et al, 2017).…”

“…Moreover, different substrates were investigated (medrane and progesterone, respectively), which could affect the influence of cytochrome b 5 on the performance of P450 (Bart & Scott, 2017). Finally, in the experiments described by Wang et al (2017), excessive concentrations of cytochrome b 5 (1:300) were used, which may lead to the contrary effect as observed in our studies, since we did not exceed a P450 to cytochrome b 5 ratio of 1:8. (Figures 4b and 4d).…”

Cytochrome P450 research and The Journal of Biological Chemistry

“…1) (15). The molecular basis for deficiencies of several P450s has been demonstrated in several inherited diseases, particularly in endocrine dysfunction (107,108). JBC has also published many of the studies on the relationship of arachidonic acid oxidation products and hypertension (Fig.…”

“…We now understand the molecular basis of many diseases, perhaps most notably in endocrine diseases (59). For instance, many clinical cases of congenital adrenal hyperplasia are now understood to result from amino acid substitutions in the CYP21A2 gene (108).…”

“…For instance, can we really understand inherited P450 deficiencies seen in the clinic at a molecular level? Are WT structures really that predictive of problems due to a single base change (108,113)?…”