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Cited by 69Hormonal Regulation and HypertensionSteroid Chemistry and BiochemistryAdrenal Hormones and Disorders

Conferring aldosterone synthesis to human CYP11B1 by replacing key amino acid residues with CYP11B2-specific ones

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Böttner, Benjamin, Denner, Karsten, Bernhardt, Rita·1998·European Journal of Biochemistry
DOI 10.1046/j.1432-1327.1998.2520458.x
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Abstract

Performing residue‐swapping experiments between the highly conserved human steroidogenic proteins CYP11B1 and CYP11B2 we recently demonstrated that replacement of specific residues at position 301, 302 and 320 in the aldosterone‐producing CYP11B2 protein for such residues that were specific for the highly similar cortisol‐producing CYP11B1 protein elevated the 11β‐hydroxylase activity dramatically. Conversely, aldosterone synthesis in the triple mutant was severely impaired. Here we provide evidence that in a reciprocal experiment, CYP11B2‐specific amino acids at position 320 and 335 endowed CYP11B1 with an 18‐oxidase function amounting to 20 % of the CYP11B2 wild‐type activity, thus changing the specificity of steroid hydroxylation by only one point mutation. Combining substitutions at positions 296, 301, 302, 320, 335 and 339 did, however, not result in further enhancement. Paradoxically, 11β‐hydroxylation was not or only marginally affected in CYP11B1 mutants, indicating an alternative structural basis for this activity in CYP11B1 compared with the engineered CYP11B2 variant. Our results suggest that the sequence spanned by amino acids 301 and 335 constitutes part of the substrate‐binding site in CYP11B1 and CYP11B2 as well. By constructing chimeric proteins we further investigated the effect of the C‐terminal portions of both proteins and found that diverging residues at positions 471, 472, 492, 493 and 494 were insignificant for the stereospecificity and regiospecificity of steroid hydroxylation.

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  • Residues 288, 296, 301, 302, 325, and, perhaps most importantly, 320 are critical for 18-oxygenase activity.

    “Residues 288, 296, 301, 302, 325, and, perhaps most importantly, 320 are critical (496, 497).”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [496]

    The Discussion (page unavailable) reports that substitutions at position 320 produced 18-oxidation activity and discusses a possible role for residue 288. It tested positions 296, 301, and 302, but does not establish each as critical for 18-oxidation; the other position it identifies with 18-oxidation activity is 335, not the claimed 325. (LLM, conf=0.87)

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Authors
Böttner, B., Denner, K., & Bernhardt, R.
Affiliations
Max Delbrück Center (DE)
ORCID
Bernhardt, Rita
Venue
European Journal of Biochemistry
Publisher
Wiley
Published
1998-03-15
Volume
252
Issue
3
Pages
458-466
ISSN
  • 0014-2956 (ISSN-L)
  • 1432-1033
DOI
10.1046/j.1432-1327.1998.2520458.x
OpenAlex
W2028238691
Semantic Scholar
a9be478e98474ed7f4fd963e1952b904296c8a05
Cited by
62
References
0
Citations by year
2024: 2 · 2023: 1 · 2021: 3 · 2020: 1 · 2018: 1 · 2017: 1 · 2016: 1 · 2015: 2 · 2014: 1 · 2013: 4 · 2012: 1
FWCI
2.01
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 1381 d)
Locations
  • European Journal of Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal Regulation and Hypertension
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
Keywords
CYP11B1, CYP11B2, aldosterone biosynthesis, substrate binding site, fusion protein, steroidogenic enzymes, regiospecificity
MeSH terms
Aldosterone, Amino Acid Sequence, Animals, Binding Sites, Humans, Hydroxylation, Molecular Sequence Data, Recombinant Fusion Proteins, Steroid 11-beta-Hydroxylase, Transfection, Mutagenesis, Site-Directed, Sequence Alignment, Sequence Deletion, Sequence Homology, Amino Acid, Protein Structure, Secondary, DNA Primers, Cytochrome P-450 CYP11B2, COS Cells, Amino Acid Substitution

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