5β-Reduced steroids and human Δ(4)-3-ketosteroid 5β-reductase (AKR1D1)

Abstract

5β-Reduced steroids are non-planar steroids that have a 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ(4)-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldo-keto reductase superfamily, but possesses an altered catalytic tetrad, in which Glu120 replaces the conserved His residue. This predominant liver enzyme generates all 5β-dihydrosteroids in the C19-C27 steroid series. Mutations exist in the AKR1D1 gene, which result in loss of protein stability and are causative in bile-acid deficiency.

Keywords: Bile acid biosynthesis; Enzyme mechanism; Genetics; Steroid metabolism.

Figure 1

Structures of biologically active 5β-reduced sterols and steroids.

Figure 2

AKR1D1 gene structures and splice variants. The nine exons are represented as filled boxes and numbered. The constitutive exons are shown in black and the alternatively spliced exons are in red.

Figure 3

Bile acid biosynthesis. AKR1D1 associated bile acid deficiency causes accumulation of Δ⁴- and allo-bile acids and prevents feedback inhibition dependent upon the primary bile acids, cholic acid and chenodeoxycholic acid. CYP7A1, cholesterol 7α-hydroxylase; CYP8B1 sterol 12α-hydroxylase; HSD3B7, 3β-hydroxysteroid dehydrogenase. Enzymes are italicized as their gene names.

Figure 4

Ordered bi bi kinetic mechanism for AKR1D1. (S) = substrate, and (P) = product.

Figure 5

Crystal Structure of AKR1D1 showing normal (A, PDB: 3CMF) and non-productive (B, PDB: 3BUR) binding modes. The steroids and NADP+ are colored in black. All the other atoms are color-coded as follows: carbon, green; oxygen, red; and nitrogen, blue; phosphor, orange. Hydrogen bonds are indicated by red dashes. All structural figures are prepared using The PyMOL Molecular Graphics System, Version 1.20 Schrödinger, LLC.

Figure 6

Crystal Structure of the AKR1D1-Glu120His mutant (magenta) in superposition with WT AKR1D1 (A, green, PDB: 3CMF) and 3α-hydroxysteroid dehydrogenase AKR1C9 (B, blue, PDB: 1AFS). In the AKR1D1 Glu120His mutant structure, the face of the steroid that presents to the cofactor is flipped and the C3 ketone of the steroid does not penetrate as deeply into the active site permitting 3β-hydroxysteroid dehydrogenase activity.

Figure 7

The chemical mechanism of AKR1D1. Note the steroid is drawn with the α-face towards the viewer and the hydride is transferred to the β-face. The tetrad residues and steroid are colored in black. All the other atoms are color-coded as in Figure 5. Hydrogen bonds are indicated by red dashes.