Structure of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme

Abstract

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21-NADPH binary complex was determined from an orthorhombic crystal belonging to space group P2(1)2(1)2(1) at a resolution of 1.8 A. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3alpha-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme-coenzyme-substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site.

Figure 1

Chemical structures of the three steroid molecules that were docked into the active site of AKR1C21. The C13 and C17 atoms are labelled. H atoms and substituted groups that are oriented towards the α-side of the steroid molecule are indicated by broken bonds and those oriented towards the β-side are indicated by solid bonds.

Figure 2

Difference electron-density map (F _o − F _c) corresponding to MPD in the active site of AKR1C21 calculated at 1.8 Å resolution and contoured at the 2.5σ level. The residues surrounding the MPD molecule and the coenzyme NADPH are labelled.

Figure 3

A ribbon representation of the AKR1C21 monomer coloured according to secondary structure (α-helices are shown in cyan, loop regions in orange and β-­sheets in purple). The ligands MPD and NADPH are shown as sticks.

Figure 4

Superimposed MPD molecules from the orthorhombic model (green) and the monoclinic model (orange) within the binding cavity of AKR1C21. The active-site water molecule is shown in blue, MPD, His117, Tyr118 and NADPH from the orthorhombic model are shown in green and MPD and 1,2-ethanediol from the monoclinic model are shown in orange. Hydrogen bonds and the distance from C4 of the nicotinamide are represented by dotted lines, with the respective distances shown.

Figure 5

Models of the three substrates (a) HPD, (b) 5β-pregnane-3α,20α-diol and (c) androstenedione docked in the active site of AKR1C21. The cofactor NADP(H) and the active-site residues are labelled. Hydrogen bonds and distances from C4 of the nicotinamide are represented by dotted lines (purple), with the respective distances shown in black.