Skip to main content
SpringerLink
Log in

The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

Abstract

Congenital adrenal hyperplasia (CAH) [OMIM 201910] is a group of autosomal recessive disorders, caused in 90–95% of cases by a deficiency of steroid 21-hydroxylase due to mutations in the CYP21A2 gene. The functional and structural effects of a novel rare missense mutation (E351K) in CYP21A2 found in a male patient with simple virilizing CAH were studied. The novel E351K point mutation is located in the ERR triad of the 21-hydroxylase. The ERR triad is a glutamine–arginine–arginine motif conserved in all cytochrome P450 sequences. The glutamate and first arginine residue are invariant in all P450 cytochrome enzymes, whereas the second arginine residue is present as arginine, histidine, or asparagine. Although the ERR triad is involved in some way to heme binding by the cytochrome P450 monooxygenases, the E351K mutation leads to severe but not complete loss of CYP21 enzyme activity. The functional analysis in COS-7 cells revealed a reduced conversion of 17-hydroxyprogesterone to 11-deoxycortisol of 1.1±0.5% (SD) and of progesterone to 11-deoxycorticosterone of 1.2±0.3% of wild-type activity. Analyzing the artificial mutants (E351D, E351I) of the E351 residue did not show a restoration of the in vitro 21-hydroxylase activity. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. The combination of in vitro enzyme function and computerized protein analysis of the E351 residue of the CYP21 protein provides experimental evidence for the ERR triad being a fundamental structural element of cytochrome P450 enzymes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Therrell BL Jr, Berenbaum SA, Manter-Kapanke V, Simmank J, Korman K, Prentice L, Gonzalez J, Gunn S (1998) Results of screening 1.9 million Texas newborns for 21-hydroxylase-deficient congenital adrenal hyperplasia. Pediatrics 101:583–590

    Google Scholar 

  2. Donohoue PA, Parker KL, Migeon CJ (2001) Congenital adrenal hyperplasia. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease, vol 3, 8th edn. McGraw-Hill, New York, pp 4077–4116

    Google Scholar 

  3. Speiser PW, White PC (2003) Congenital adrenal hyperplasia. N Engl J Med 349:776–788

    Google Scholar 

  4. Carroll MC, Campbell RD, Porter RR (1985) Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man. Proc Natl Acad Sci USA 82:521–525

    Google Scholar 

  5. White PC, Grossberger D, Onufer BJ, Chaplin DD, New MI, Dupont B, Strominger JL (1985) Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci USA 82:1089–1093

    Google Scholar 

  6. White PC, New MI, Dupont B (1986) Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci USA 83:5111–5115

    Google Scholar 

  7. Higashi Y, Yoshioka H, Yamane M, Gotoh O, Fujii-Kuriyama Y (1986) Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene. Proc Natl Acad Sci USA 83:2841–2845

    Google Scholar 

  8. Koppens PF, Hoogenboezem T, Degenhart HJ (2002) Carriership of a defective tenascin-X gene in steroid 21-hydroxylase deficiency patients: TNXB -TNXA hybrids in apparent large-scale gene conversions. Hum Mol Genet 11:2581–2590

    Google Scholar 

  9. The Human Gene Mutation Database, available at: http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/120605.html

  10. Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna MT, Lesser M, New MI, White PC (1992) Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 90:584–595

    Google Scholar 

  11. Wedell A, Thilen A, Ritzen EM, Stengler B, Luthman H (1994) Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 78:1145–1152

    Google Scholar 

  12. Wilson RC, Mercado AB, Cheng KC, New MI (1995) Steroid 21-hydroxylase deficiency: genotype may not predict phenotype. J Clin Endocrinol Metab 80:2322–2329

    Google Scholar 

  13. Ezquieta B, Oliver A, Gracia R, Gancedo PG (1995) Analysis of steroid 21-hydroxylase gene mutations in the Spanish population. Hum Genet 96:198–204

    Google Scholar 

  14. Jaaskelainen J, Levo A, Voutilainen R, Partanen J (1997) Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: good correlation in a well defined population. J Clin Endocrinol Metab 82:3293–3297

    Google Scholar 

  15. Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP (2000) Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 85:1059–1065

    Google Scholar 

  16. Baumgartner-Parzer SM, Schulze E, Waldhausl W, Pauschenwein S, Rondot S, Nowotny P, Meyer K, Frisch H, Waldhauser F, Vierhapper H (2001) Mutational spectrum of the steroid 21-hydroxylase gene in Austria: identification of a novel missense mutation. J Clin Endocrinol Metab 86:4771–4775

    Google Scholar 

  17. Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, Hermus AR, Sistermans EA (2003) CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations. J Clin Endocrinol Metab 88:3852–3859

    Google Scholar 

  18. Kharrat M, Tardy V, M’Rad R, Maazoul F, Jemaa LB, Refai M, Morel Y, Chaabouni H (2004) Molecular genetic analysis of Tunisian patients with a classic form of 21-hydroxylase deficiency: identification of four novel mutations and high prevalence of Q318X mutation. J Clin Endocrinol Metab 89:368–374

    Google Scholar 

  19. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J (1995) Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3:41–62

    Google Scholar 

  20. Antonarakis SE (1998) Recommendations for a nomenclature system for human gene mutations. Nomenclature working group. Hum Mutat 11:1–3

    Google Scholar 

  21. Krone N, Roscher AA, Schwarz HP, Braun A (1998) Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency. Clin Chem 44:2075–2082

    Google Scholar 

  22. Krone N, Braun A, Weinert S, Peter M, Roscher AA, Partsch CJ, Sippell WG (2002) Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia. Clin Chem 48:818–825

    Google Scholar 

  23. Vriend G (1990) What if: a molecular modeling and drug design program. J Mol Graph 8:52–56, (29)

    Google Scholar 

  24. Carson M (1991) Ribbons 2.0. J Appl Crystallogr 24:946–950

    Article  Google Scholar 

  25. White PC, Speiser PW (2000) Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 21:245–291

    Google Scholar 

  26. Lajic S, Robins T, Krone N, Schwarz HP, Wedell A (2001) CYP21 mutations in simple virilizing congenital adrenal hyperplasia. J Mol Med 79:581–586

    Google Scholar 

  27. Lajic S, Clauin S, Robins T, Vexiau P, Blanche H, Bellanne-Chantelot C, Wedell A (2002) Novel mutations in CYP21 detected in individuals with hyperandrogenism. J Clin Endocrinol Metab 87:2824–2829

    Google Scholar 

  28. Nunez BS, Lobato MN, White PC, Meseguer A (1999) Functional analysis of four CYP21 mutations from Spanish patients with congenital adrenal hyperplasia. Biochem Biophys Res Commun 262:635–637

    Google Scholar 

  29. Mornet E, Gibrat JF (2000) A 3D model of human P450c21: study of the putative effects of steroid 21-hydroxylase gene mutations. Hum Genet 106:330–339

    Google Scholar 

  30. Yoshikawa K, Noguti T, Tsujimura M, Koga H, Yasukochi T, Horiuchi T, Go M (1992) Hydrogen bond network of cytochrome P-450cam: a network connecting the heme group with helix K. Biochim Biophys Acta 1122:41–44

    Google Scholar 

  31. Kitamura M, Buczko E, Dufau ML (1991) Dissociation of hydroxylase and lyase activities by site-directed mutagenesis of the rat P45017 alpha. Mol Endocrinol 5:1373–1380

    Google Scholar 

  32. Shimizu T, Tateishi T, Hatano M, Fujii-Kuriyama Y (1991) Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase. J Biol Chem 266:3372–3375

    Google Scholar 

  33. Furuya H, Shimizu T, Hirano K, Hatano M, Fujii-Kuriyama Y, Raag R, Poulos TL (1989) Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin. Biochemistry 28:6848–6857

    Google Scholar 

  34. Wester MR, Johnson EF, Marques-Soares C, Dansette PM, Mansuy D, Stout CD (2003) Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes. Biochemistry 42:6370–6379

    Google Scholar 

  35. Lajic S, Levo A, Nikoshkov A, Lundberg Y, Partanen J, Wedell A (1997) A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction. Hum Genet 99:704–709

    Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. W.L. Miller (Dept. of Pediatrics, University of California, San Francisco, Calif., USA) for providing the CYP21A2 cDNA and the anti-human-CYP21 rabbit polyclonal antiserum. We appreciate the expert technical assistance of Gisela Hohmann and Brigitte Andresen and thank Joanna Voerste for linguistic help with the manuscript.

Author information

Authors and Affiliations

  1. Division of Pediatric Endocrinology, Department of Pediatrics, Christian-Albrechts-Universität zu Kiel, Universitätsklinikum Schleswig-Holstein (Campus Kiel), Schwanenweg 20, 24105, Kiel, Germany

    Nils Krone, Felix G. Riepe, Carl-Joachim Partsch & Wolfgang G. Sippell

  2. Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstrasse 40, 24098, Kiel, Germany

    Joachim Grötzinger

  3. Department of Pediatrics, Westfälische Wilhelms-Universität Münster, Albert-Schweizer-Strasse 33, 48149, Münster, Germany

    Jürgen Brämswig

Authors
  1. Nils Krone
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Felix G. Riepe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joachim Grötzinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carl-Joachim Partsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jürgen Brämswig
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wolfgang G. Sippell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nils Krone.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krone, N., Riepe, F.G., Grötzinger, J. et al. The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions. J Mol Med 83, 561–568 (2005). https://doi.org/10.1007/s00109-005-0655-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-005-0655-3

Keywords

Search

Navigation