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Mammalian steroid 5β-reductases belong to the Aldo-Keto Reductase 1D sub-family and are essential for the formation of A-ring 5β-reduced steroids. Steroid 5β-reduction is required for the biosynthesis of bile-acids and the metabolism of all steroid hormones that contain a Δ4-3-ketosteroid functionally to yield the 5β-reduced metabolites. In mammalian AKR1D enzymes the conserved catalytic tetrad found in all AKRs (Y55, H117, K84 and D50) has changed in that the conserved H117 is replaced with a glutamic acid (E120). E120 may act as a "superacid" to facilitate enolization of the Δ4-ketosteroid. In addition, the absence of the bulky imidazole side chain of histidine in E120 permits the steroid to penetrate deeper into the active site so that hydride transfer can occur to the steroid C5 position. In murine steroid 5β-reductase AKR1D4, we find that there is a long-form, with an 18 amino-acid extension at the N-terminus (AKR1D4L) and a short-form (AKR1D4S), where the latter is recognized as AKR1D4 by the major data-bases. Both enzymes were purified to homogeneity and product profiling was performed. With progesterone and cortisol, AKR1D4L and AKR1D4S catalyzed smooth conversion to the 5β-dihydrosteroids. However, with Δ4-androstene-3,17-dione as substrate, a mixture of products was observed which included, 5β-androstane-3,17-dione (expected) but 3α-hydroxy-5β- androstan-17-one was also formed. The latter compound was distinguished from its isomeric 3β-hydroxy-5β-androstan-17-one by forming picolinic acid derivatives followed by LC-MS. These data show that AKR1D4L and AKR1D4S also act as 3α-hydroxysteroid dehydrogenases when presented with Δ4-androstene-3,17-dione and suggest that E120 alters the position the steroid to enable a correct trajectory for hydride transfer and may not act as a "superacid".

Original publication




Journal article


Chem Biol Interact

Publication Date





163 - 170


Aldo-keto reductase, Hydroxysteroid dehydrogenase, Steroid 5β-reductase, Androstanes, Animals, Biocatalysis, Catalytic Domain, Chromatography, High Pressure Liquid, Glutamic Acid, Humans, Isomerism, Kinetics, Liver, Mice, Oxidation-Reduction, Oxidoreductases, Protein Isoforms, Steroids, Substrate Specificity, Tandem Mass Spectrometry