Rate of steroid double-bond reduction catalysed by the human steroid 5β-reductase (AKR1D1) is sensitive to steroid structure: implications for steroid metabolism and bile acid synthesis

Abstract

Human AKR1D1 (steroid 5β-reductase/aldo-keto reductase 1D1) catalyses the stereospecific reduction of double bonds in Δ4-3-oxosteroids, a unique reaction that introduces a 90° bend at the A/B ring fusion to yield 5β-dihydrosteroids. AKR1D1 is the only enzyme capable of steroid 5β-reduction in humans and plays critical physiological roles. In steroid hormone metabolism, AKR1D1 serves mainly to inactivate the major classes of steroid hormones. AKR1D1 also catalyses key steps of the biosynthetic pathway of bile acids, which regulate lipid emulsification and cholesterol homoeostasis. Interestingly, AKR1D1 displayed a 20-fold variation in the kcat values, with steroid hormone substrates (e.g. aldosterone, testosterone and cortisone) having significantly higher kcat values than steroids with longer side chains (e.g. 7α-hydroxycholestenone, a bile acid precursor). Transient kinetic analysis revealed striking variations up to two orders of magnitude in the rate of the chemistry step (kchem), which resulted in different rate determining steps for the fast and slow substrates. By contrast, similar Kd values were observed for representative fast and slow substrates, suggesting similar rates of release for different steroid products. The release of NADP+ was shown to control the overall turnover for fast substrates, but not for slow substrates. Despite having high kchem values with steroid hormones, the kinetic control of AKR1D1 is consistent with the enzyme catalysing the slowest step in the catabolic sequence of steroid hormone transformation in the liver. The inherent slowness of the conversion of the bile acid precursor by AKR1D1 is also indicative of a regulatory role in bile acid synthesis.

Figure 1. Biological role of AKR1D1 (A and B) and chemical structures of the steroid substrates tested

(A) Steroid hormones are metabolized by the concerted actions of AKR1D1 and AKR1C enzymes. S, steroid hormone. (B) AKR1D1 is responsible for the 5β-reduction in all pathways for the synthesis of primary bile acids cholic acid and chenodeoxycholic acid. R₁, -H or -OH; R₂, -CH₃ or -COOH. (C) All substrates are similar in chemical structure and have an identical A-ring, where the reaction occurs.

Figure 2. Macroscopic kinetic events in AKR1D1 catalysis

The turnover number (k_cat) is defined by the rate constants of three steps: the chemistry step (k_chem), the release of steroid product (k_r,Sp) and the release of NADP⁺ (k _r,NADP ⁺). E, enzyme; S_S, steroid substrate; S_P, steroid product.

Figure 3. Representative kinetic traces for the multiple-turnover reactions catalysed by AKR1D1 in the transient state

(A) Averaged progress curves of decreases in NADPH fluorescence for samples containing testosterone (red), cortisone (blue) and aldosterone (green). (B) Averaged progress curves of decreases in NADPH fluorescence for samples containing no steroid (black), cholestenone (magenta) or 7α-hydroxycholestenone (cyan). (C) Fitting of an averaged trace of cortisone multiple-turnover reaction to the burst equation. (D) Fitting of an averaged trace of 7α-hydroxycholestenone multiple-turnover reaction to the linear equation. The initial increase in fluorescence signal in the reaction of 7α-hydroxycholestenone was also observed in the sample containing no steroid and was not used in data fitting. Fitted lines are in grey and are mostly superimposed by the actual data points. Residual plots demonstrate the quality of the fit.

Figure 4. Representative kinetic traces for the single-turnover reactions catalysed by AKR1D1

(A) The averaged progress curves for the reaction of the pre-mixed enzyme–NADPH solution with cortisone. Data were fitted to a double-exponential function. (B) The averaged progress curves for the reaction of the pre-mixed enzyme–NADPH solution with 7α-hydroxycholestenone. Data were fitted to a single-exponential function. Fitted lines are shown in grey. Residual plots demonstrate the quality of the fit. Note the different time window used for the two substrates.

Figure 5. Determination of cortisone dissociation constant (K_d) by fluorescence titration

Formation of the AKR1D1–NADPH complex quenches intrinsic protein fluorescence and generates an energy transfer band at 460 nm. Addition of steroid quenches the energy transfer band, which was used to titrate the AKR1D1–NADPH–steroid ternary complex.

Figure 6. Representative kinetic traces from competition of cofactor binding

The averaged progress curve of the energy transfer fluorescence signal observed upon mixing an enzyme solution with excess NADPH solution. The final sample contained 0.5 μM AKR1D1 and 50 μM NADPH (□).The averaged progress curve observed upon mixing an enzyme–NADP ⁺ solution with excess NADPH solution. The final sample contained 0.5 μM AKR1D1, 2.5 μM NADP ⁺ and 50 μM NADPH (×). The averaged progress curve observed upon rapid mixing an NADP ⁺ solution with excess NADPH solution. The final sample contained 2.5 μM NADP ⁺ and 50 μM NADPH (○). Data for the E (enzyme) + NADPH line (□) were fitted to a double-exponential function, whereas data for E NADP ⁺ + NADPH line were fitted to a single-exponential function (×). The fitted lines are shown in grey.