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

Abstract

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 systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ(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 5β-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.

Figure 1

Substrates and inhibitors evaluated.

Figure 2

pH stability and pH-rate optimal of AKR1D1. pH stability (■) and pH optima (▲) of the enzyme was tested from pH 5 to 9 using 10 μM testosterone.

Figure 3

The active site of AKR1D1 showing (A) the surface of the cylindrical cavity when cortisone is bound (PDB ID: 3CMF). The steroid A ring is buried in the active site, while the C17 side chain protrudes out of the cavity. The surface is colored according to hydrophobicity (yellow corresponds to hydrophobic residues; and blue corresponds to hydrophilic residues). (B) The residues defining the nonproductive binding pocket for testosterone are shown (PDB ID: 3BUR).