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 292 (26): 10767-10778

PMID: 28539365 · PMCID: PMC5491764 · DOI:10.1074/jbc.M117.792465

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 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 (17α-OH-progesterone). We found that the 17α-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 () for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome , 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--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 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.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (11)

Adrenal Hyperplasia, Congenital Circular Dichroism Cytochromes b5 Deuterium Exchange Measurement Enzyme Stability Hot Temperature Humans Mutation Protein Domains Spectrophotometry, Ultraviolet Steroid 21-Hydroxylase

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