Substrate specificity and kinetic mechanism of 3β-hydroxy-Δ5-C27-steroid oxidoreductase

Abstract

Cholesterol is a key sterol whose homeostasis is primarily maintained through bile acid metabolism. Proper bile acid formation is vital for nutrient and fat-soluble vitamin absorption and emulsification of lipids. Synthesis of bile acids occurs through two main pathways, both of which rely on 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase (HSD3B7) to begin epimerization of the 3β hydroxyl of cholesterol into its active 3α conformation. In this sequence, HSD3B7 catalyzes the dehydrogenation of the 3β-hydroxy group followed by isomerization of the Δ⁵-cholestene-3-one. These reactions are some of the many steps that transform cholesterol for either storage or secretion. HSD3B7 has distinct activity from other 3β-HSD family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. In addition, the role of HSD3B7 in health and disease positions it as a metabolic vulnerability that could be harnessed as a therapeutic target. To this end, we evaluated the mechanism of HSD3B7 catalysis and reveal that HSD3B7 displays activity toward diverse 7α-hydroxylated oxysterols. HSD3B7 retains its catalytic efficiency toward these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Experiments aimed at determining substrate order are consistent with HSD3B7 catalyzing a sequential ordered bi-bi reaction mechanism with the binding of NAD⁺ followed by 7α-hydroxycholesterol to form a central complex. HSD3B7 bifunctional activity is dependent on membrane localization through a putative membrane-associated helix giving insight into potential regulation of enzyme activity. We found strong binding of the NADH product thought to activate the isomerization reaction. Homology models of HSD3B7 reveal a potential substrate pocket that allows for oxysterol binding, and mutagenesis was utilized to support this model. Together, these studies offer an understanding of substrate specificity and kinetic mechanism of HSD3B7, which can be exploited for future drug development.

Keywords: bile acid synthesis; cholesterol metabolism; enzyme mechanism; membrane protein; metabolism; oxidoreductase.

Figure 1

Kinetic characterization of HSD3B7 for its 7α-oxysterol substrates and NAD+.A, structure of 7α-oxysterol substrates used in this study with varying modifications highlighted in red. B, steady-state enzyme kinetics for oxysterol substrates and HSD3B7 with constant NAD⁺. C, steady-state enzyme kinetics for NAD⁺ and HSD3B7 with constant oxysterol substrate. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase.

Figure 2

7α-oxysterol specificity of HSD3B7.A, structures of 20(S) and 24(S)-hydroxycholesterol. B, activity assay of WT HSD3B7 in the presence of 7α-OHC, 20(S)-hydroxycholesterol, and 24(S)-hydroxycholesterol. The axis is adjusted to show low activity toward non-7α containing oxysterols. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase.

Figure 3

Mechanism of HSD3B7 substrate binding.A, relative activity of catalytic mutant K163R. B, MST relative fluorescence traces of HSD3B7 in the presence (red) and absence (black) of 150 μM 7-HCO. C, MST relative fluorescence after 20 s in the presence (red) or absence (black) of 150 μM 7α-OHC. D, MST relative fluorescence traces of K163R with 1 mM NAD⁺ in the presence (red) and absence (black) of 150 μM 7α-OHC. E, MST relative fluorescence after 20 s of HSD3B7 with 1 mM NAD⁺ in the presence (red) or absence (black) of 150 μM 7-HCO. F, dehydrogenase scheme in Cleland representation. Order of product release was not evaluated. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase; MST, microscale thermophoresis.

Figure 4

Fluorescence emission spectra of HSD3B7 and titrated NADH.A, HSD3B7 reaction mechanism for 3β-HSD and isomerase activity. B, emission traces of varying NADH (0.15 μM–10 μM) in the presence of 1.8 μM HSD3B7 after excitation of the intrinsic tryptophan fluorescence. C, change in fluorescence at 460 nm versus NADH concentration fit to the Morrison equation. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase.

Figure 5

Effect of membrane-associated helix (MAH) on HSD3B7 catalysis.A, domain architecture of WT HSD3B7 and 3β-HSD domain with deleted MAH and transmembrane helix hidden Markov model (TMHMM) for HSD3B7 sequence. Cytoplasmic, noncytoplasmic, and membrane-associated predicted sequences are represented by blue, gray, and red, respectively. B, second construct replaces the MAH with a GGGS linker and TMHMM for HSD3B7 sequence. Cytoplasmic, noncytoplasmic, and membrane-associated predicted sequences are represented by black, gray, and red, respectively. C, mass photometry of ΔMAH construct with a predicted molecular weight of 38 kD. D, thermostability of ΔMAH in the presence and absence of substrates. T_m of cHSD3B7-ΔMAH, cHSD3B7-ΔMAH + 7α-OHC, and cHSD3B7-ΔMAH + NAD⁺ is 46.4 ± 0.2, 47.5 ± 0.02, and 47.4 ± 0.2 °C, respectively. E, steady-state kinetics of ΔMAH in the presence of excess NAD⁺. F, steady-state kinetics of ΔMAH in the presence of excess 7α-OHC. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase; 7α-OHC, 7α-hydroxycholesterol.

Figure 6

Alphafold modeling and molecular docking of HSD3B7.A, WT HSD3B7 model with docking of substrates NAD⁺ and 7α-OHC. OPM membrane prediction is show in red, with HSD3B7 on the cytosolic side of the membrane. B, Surface rendering of WT HSD3B7 model depicting oxysterol pocket, with 7α-OHC docked within the pocket. C, docking pose of 7α-OHC within a predicted binding pocket with key interactions labeled. D, docking pose of 7α,27-diHC within a predicted binding pocket with key interactions labeled. E, Y-S-N-K catalytic tetrad of HSD3B7 in docking model of WT HSD3B7 with relative positioning to NAD⁺ and 7α-OHC. F, overlay of WT HSD3B7 and cHSD3B7-ΔMAH models. Rotated models show differences in access to the oxysterol binding pocket.HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase; 7α-OHC, 7α-hydroxycholesterol.

Figure 7

HSD3B7 mutant activity and steady-state kinetics.A, relative activity of HSD3B7 predicted oxysterol binding pocket mutants. B, steady-state kinetics of F205A mutant in the presence of excess NAD⁺. C, steady-state kinetics of F205A mutant in the presence of excess 7α-OHC. D, F93A mutant activity in the presence of excess NAD⁺. HSD3B7, 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase; 7α-OHC, 7α-hydroxycholesterol.