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

Journal Article · · Journal of Biological Chemistry
 [1];  [1];  [1];  [1];  [2];  [1];  [1];  [1]
  1. Vanderbilt Univ., Nashville, TN (United States)
  2. Univ. of Texas at San Antonio, TX (United States)
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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 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 (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 (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.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (US). Advanced Photon Source (APS)
Sponsoring Organization:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI ID:
1373792
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 26 Vol. 292; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Cited By (7)

Identification and circumvention of bottlenecks in CYP21A2‐mediated premedrol production using recombinant Escherichia coli journal December 2019
Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae journal August 2019
Detection of a novel severe mutation affecting the CYP21A2 gene in a Chilean male with salt wasting congenital adrenal hyperplasia journal September 2019
Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes journal December 2017
Cytochrome P450 research and The Journal of Biological Chemistry journal February 2019
Phenotype heterogeneity of congenital adrenal hyperplasia due to genetic mosaicism and concomitant nephrogenic diabetes insipidus in a sibling journal July 2018
Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli collection January 2019

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
X-ray crystallography
circular dichroism (CD)
congenital adrenal hyperplasia
cytochrome P450
isotope effect
steroid
steroid biosynthesis
steroid hormone