The effect of disease associated point mutations on 5β-reductase (AKR1D1) enzyme function
Open accessAbstract
The stereospecific 5β-reduction of Δ(4)-3-ketosterols is very difficult to achieve chemically and introduces a 90° bend between ring A and B of the planar steroid. In mammals, the reaction is catalyzed by steroid 5β-reductase, a member of the aldo-keto reductase (AKR) family. The human enzyme, AKR1D1, plays an essential role in bile-acid biosynthesis since the 5β-configuration is required for the emulsifying properties of bile. Deficient 5β-reductase activity can lead to cholestasis and neo-natal liver failure and is often lethal if it remains untreated. In five patients with 5β-reductase deficiency, sequencing revealed individual, non-synonymous point mutations in the AKR1D1 gene: L106F, P133R, G223E, P198L and R261C. However, mapping these mutations to the AKR1D1 crystal structure failed to reveal any obvious involvement in substrate or cofactor binding or catalytic mechanism, and it remained unclear whether these mutations could be causal for the observed disease. We analyzed the positions of the reported mutations and found that they reside in highly conserved portions of AKR1D1 and hypothesized that they would likely lead to changes in protein folding, and hence enzyme activity. Attempts to purify the mutant enzymes for further characterization by over-expression in Escherichia coli yielded sufficient amounts of only one mutant (P133R). This enzyme exhibited reduced K(m) and k(cat) values with the bile acid intermediate Δ(4)-cholesten-7α-ol-3-one as substrate reminiscent of uncompetitive inhibition. In addition, P133R displayed no change in cofactor affinity but was more thermolabile as judged by CD-spectroscopy. When all AKR1D1 mutants were expressed in HEK 293 cells, protein expression levels and enzyme activity were dramatically reduced. Furthermore, cycloheximide treatment revealed decreased stability of several of the mutants compared to wild type. Our data show, that all five mutations identified in patients with functional bile acid deficiency strongly affected AKR1D1 enzyme functionality and therefore may be causal for this disease.
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The deficiency is exacerbated by inability to form sufficient bile acids to feedback-inhibit CYP7A1, leading to further elevation of allo-bile acids.
“This deficiency is exacerbated by the inability to from sufficient bile acids to feed-back and inhibit the rate determining step in the pathway, 7α-hydroxylase (CYP7A1), and thus the formation of allo-bile acids will be further elevated [95–99].”
From Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism (2014) · cites this paper as [98]
Inherited AKR1D1 mutations cause bile-acid deficiency in infants characterized by reduced bile acid formation and accumulation of hepatotoxic Δ4-3-keto and 5α-reduced allo-bile acids.
“Inherited mutations in AKR1D1 have been studied in patients with bile-acid deficiency that develops in infants and is characterized by reduced formation of bile acids and accumulation of Δ4-3-keto and 5α-reduced (allo-bile acids) which are hepatotoxic.”
From Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism (2014) · cites this paper as [98]
The mutants showed lower 5β-reductase activity than wild type, in agreement with clinical observations of bile acid deficiency.
“The mutants thus showed lower 5β-reductase activity as compared to wild type which is in agreement with clinical observations of bile acid deficiency [98, 101].”
From Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism (2014) · cites this paper as [98]
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- Authors
- Mindnich, R., Drury, J. E., & Penning, T. M.
- Affiliations
- University of Pennsylvania (US)
- Corresponding author
- Penning, Trevor M.
- ORCID
- Penning, Trevor M.
- Venue
- Chemico-Biological Interactions
- Publisher
- Elsevier BV
- Published
- 2011-05
- Volume
- 191
- Issue
- 1-3
- Pages
- 250-254
- ISSN
- 0009-2797 (ISSN-L)
- PMC
- PMC3101292
- OpenAlex
- W2093795670
- Cited by
- 16
- References
- 32
- Citations by year
- 2024: 1 · 2021: 1 · 2019: 2 · 2018: 2 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 3 · 2012: 1
- FWCI
- 0.56
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Chemico-Biological Interactions (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute on Alcohol Abuse and Alcoholism (R13AA019612)
- National Cancer Institute (R13CA097985)
- National Institute of Diabetes and Digestive and Kidney Diseases (5-R01-DK047015-13, R01DK047015)
- National Institute of Environmental Health Sciences (P30ES013508)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Aldose Reductase and Taurine
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
- Keywords
- AKR1D1, aldo-keto reductase, point mutations, bile acid deficiency, cholestasis, neonatal liver failure, enzyme kinetics, uncompetitive inhibition, protein stability, protein folding, pathogenic variants
- MeSH terms
- Animals, Bile Acids and Salts, Disease, Humans, Oxidoreductases, Conserved Sequence, Point Mutation, HEK293 Cells
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