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

Abstract

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 thatmore » 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.« less

Authors:
 [1];  [1];  [1];  [1];  [2];  [1];  [1]; ORCiD logo [1]
  1. Vanderbilt Univ., Nashville, TN (United States)
  2. Univ. of Texas at San Antonio, TX (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1373792
Grant/Contract Number:  
R01 GM103937
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 26; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; circular dichroism (CD); cytochrome P450; isotope effect; steroid hormone; X-ray crystallography; congenital adrenal hyperplasia; steroid; steroid biosynthesis

Citation Formats

Wang, Chunxue, Pallan, Pradeep S., Zhang, Wei, Lei, Li, Yoshimoto, Francis K., Waterman, Michael R., Egli, Martin, and Guengerich, F. Peter. Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia. United States: N. p., 2017. Web. doi:10.1074/jbc.m117.792465.
Wang, Chunxue, Pallan, Pradeep S., Zhang, Wei, Lei, Li, Yoshimoto, Francis K., Waterman, Michael R., Egli, Martin, & Guengerich, F. Peter. Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia. United States. https://doi.org/10.1074/jbc.m117.792465
Wang, Chunxue, Pallan, Pradeep S., Zhang, Wei, Lei, Li, Yoshimoto, Francis K., Waterman, Michael R., Egli, Martin, and Guengerich, F. Peter. 2017. "Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia". United States. https://doi.org/10.1074/jbc.m117.792465. https://www.osti.gov/servlets/purl/1373792.
@article{osti_1373792,
title = {Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia},
author = {Wang, Chunxue and Pallan, Pradeep S. and Zhang, Wei and Lei, Li and Yoshimoto, Francis K. and Waterman, Michael R. and Egli, Martin and Guengerich, F. Peter},
abstractNote = {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.},
doi = {10.1074/jbc.m117.792465},
url = {https://www.osti.gov/biblio/1373792}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 26,
volume = 292,
place = {United States},
year = {Fri Jun 30 00:00:00 EDT 2017},
month = {Fri Jun 30 00:00:00 EDT 2017}
}

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