Biochemical characterization of the bile acid synthetic enzyme 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII

Abstract

Cholesterol is a key biological molecule that is a vital precursor of steroid molecule biosynthesis, including steroid hormones, bile acids and vitamin D3.These processes require 3β‐hydroxysteroid dehydrogenase (3β‐HSD) enzymes, which catalyze dehydrogenation of hydroxysteroids. This family of enzymes catalyzes a wide variety of reaction mechanisms, carrying out a combination of oxidation, isomerization and decarboxylase reactions that result in the formation of cholesterol, steroid hormones and bile acids. 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII (HSD3B7) is responsible for the NAD+ dependent conversion of 7α‐hydroxycholesterol (7‐OHC) to 7α‐hydroxy‐3‐oxo‐4‐cholestenoate (7‐HOC), the second step of the bile acid synthesis pathway, which is vital for nutrient absorption and excretion of excess lipids. This oxidation reaction is one of many steps that increases the solubility of cholesterol for either storage or secretion. HSD3B7 shows substantial functional differences from other family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. To this end, I have characterized HSD3B7 using Michaelis‐Menten kinetics, thermal shift assays, and begun x‐ray crystallographic studies. These studies reveal that HSD3B7 displays activity towards multiple bile acid precursors with differing hydrocarbon tail oxidation, with Km values in the low micromolar range. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Lastly, HSD3B7 can be crystallized after modification to its membrane associated region, which will result in one of the first structures of a 3β‐HSD enzyme. Together these studies provide key insight into the mechanism of substrate specificity of HSD3B7 and appears amenable for X‐ray crystal structure determination.