Journal of Biological Chemistry
Volume 285, Issue 32, 6 August 2010, Pages 24529-24537
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Molecular Bases of Disease
Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency*

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Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5β-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5β-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Δ4 double bond in 3-keto steroids and sterols to yield the 5β-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5β-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5β-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced Km and Vmax reminiscent of uncompetitive kinetics with 4-cholesten-7α-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5β-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.

Bile Acid
Enzyme Kinetics
Mutant
Protein Stability
Steroid
5beta-Reduction
Aldo-keto Reductase

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*

This work was supported, in whole or in part, by National Institutes of Health Grants R01-DK47015 and P30 ES013508 (to T. M. P.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1.

1

Both authors contributed equally to this work.