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Substrate specificity and inhibitor analyses of human steroid 5?-reductase (AKR1D1).

ABSTRACT: Human steroid 5?-reductase (aldo-keto reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of ?(4)-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 ?(4)-ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5?-steroid metabolites present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the C11 position was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1. By contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.

SUBMITTER: Chen M

PROVIDER: S-EPMC3056882 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Substrate specificity and inhibitor analyses of human steroid 5β-reductase (AKR1D1).

Chen Mo M Drury Jason E JE Penning Trevor M TM

Steroids 20110119 5

Human steroid 5β-reductase (aldo-keto reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of Δ(4)-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we syst ...[more]

PMID: 21255593

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Project description:Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.

Dataset Information

Substrate specificity and inhibitor analyses of human steroid 5?-reductase (AKR1D1).

Publications

Substrate specificity and inhibitor analyses of human steroid 5β-reductase (AKR1D1).

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