Functional basis of P 450 21 A 2 deficiencies 1 Functional Analysis of Human Cytochrome P 450 21 A 2 Variants Involved in Congenital Adrenal Hyperplasia

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17αhydroxy(OH)progesterone to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of wild-type (WT) human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH progesterone). We found that the 17α-OH progesteroneand progesteronebound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting (SW) or nonclassical (NC) 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 10-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 SWand NCassociated 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) 21A2, an enzyme located in the adrenal cortex, catalyzes the 21-hydroxylation of both progesterone and 17α-hydroxy (OH) progesterone, forming 11deoxycorticosterone and 11-deoxycortisol, respectively (2-4). Deficiencies in the CYP21A2 gene are common and are involved in ~ 95% 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 ~ 1/15,000 worldwide, with well over 100 different genetic variants having been diagnosed. The http://www.jbc. org/cgi/doi/10.1074/jbc.M117.792465 The latest version is at JBC Papers in Press. Published on May 24, 2017 as Manuscript M117.792465 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M ay 9, 2017 hp://w w w . jb.org/ D ow nladed from Functional basis of P450 21A2 deficiencies 2 phenotypes are generally classified as nonclassical (NC), simple virilizing (SV), and saltwasting (SW), in order of increasing severity (2, 3, 6). The great majority of mutations in CYP21A2 can be traced to the non-functional pseudogene of P450 21A2 (CYP21A1P), which is found in more than 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 17α-OH progesterone (8), which provided insight into roles of some of the residues involved in the enzymatic deficiencies. Using our bovine P450 21A2 structure, New and her associates modeled the human P450 21A2 enzyme and discussed how the low activities of some of the variants might be explained (9). We subsequently crystallized and published a structure of human P450 21A2 bound with its other substrate, progesterone (10). This structure showed some important differences with the bovine enzyme, and we discussed how these could effect the conclusions reached with modeling based on the bovine enzyme (11). However, even with the P450 21A2 structures available, it is not clear what all of the factors causing low catalytic activity are. We now report a structure of human P450 21A2 bound to its substrate 17α-OH progesterone. In addition, we expressed and purified 12 human P450 21A2 variants and measured kcat and Km parameters for 21-hydroxylation of both progesterone and 17α-OH progesterone. Large kinetic deuterium isotope effects were observed with all six of the variants examined, indicating that C-H bond breaking is a rate-limiting step in all cases. The stability of the variants was addressed in several ways. We discuss possible reasons for low activity of the variants, especially those variants not analyzed previously (11).