Structural enzymology of cholesterol biosynthesis and storage

Abstract

Cholesterol biosynthesis occurs in the endoplasmic reticulum (ER). Its lego-like construction from water-soluble small metabolites via intermediates of increasing complexity to water-insoluble cholesterol requires numerous distinct enzymes. Dysfunction of the involved enzymes can cause several human inborn defects and diseases. Here, we review recent structures of three key cholesterol biosynthetic enzymes: Squalene epoxidase (SQLE), NAD(P)-dependent steroid dehydrogenase-like (NSDHL), and 3β-hydroxysteroid Δ⁸-Δ⁷ isomerase termed EBP. Moreover, we discuss structures of acyl-CoA:cholesterol acyltransferase (ACAT) enzymes, which are responsible for forming cholesteryl esters from cholesterol to maintain cholesterol homeostasis in the ER. The structures of these enzymes reveal their catalytic mechanism and provide a molecular basis to develop drugs for treating diseases linked to their dysregulation.

Figure 1.. Biosynthetic pathway of cholesterol and cholesteryl ester.

Enzymes that have been discussed in this review are colored in red. The left lane represents the Bloch pathway; the right lane represents the Kandutsch-Russell pathway.

Figure 2.. Structural mechanisms of three key enzymes in the cholesterol biosynthetic pathway.

(a) Overall structure of the catalytic domain of SQLE bound to the inhibitor compound-4” (Cmpd-4”) (pdb: 6C6N). The FAD-binding domain, substrate-binding domain and C-terminal helical domain are colored in cyan, lime, and light pink, respectively. FAD (stick representation with carbon atoms in yellow) and Cmpd-4” (magenta) are shown. (b) The active site of SQLE. Y195 in the inhibitor-bound structure (lime) forms a hydrogen bond with the tertiary amine of Cmpd-4’’, while in the FAD-only structure (light blue, pdb: 6C6R), Y195 (light blue) interacts with Q168 (light blue) through a hydrogen bond. The black arrow indicates the conformational change of Y195. The side chains of Y335 and E165 and the main chain of I162 interact with N5 of FAD via a bridging water molecule (brown sphere). Y335, E165, and I162 are shown as sticks. (c) Overall structure of NSDHL complexed with NAD⁺ (pdb: 6JKH). NAD⁺ (pale cyan) is shown in stick representation. (d) The active site of NSDHL. The catalytic residues Y172 and K176 and motif G44-G50 are colored in green. The black arrow indicates the conformational change of loop H201-L211 from the NAD-bound structure (green and orange cartoon) to the apo structure (light pink and yellow cartoon, pdb: 6JKG). K232’ (orange) from the neighboring monomer interacts with loop H201-L211 at the dimer interface. (e) Overall structure of U18666A-bound EBP (6OHT). U18666A (magenta) is shown in stick representation. (f) The active site of EBP. The amine group of U18666A, which forms hydrophilic interactions with E122 and N193, is stabilized by W196 via a π-cation interaction. H76, E80, and E122 form a hydrogen-bonding network during enzymatic reaction. Residues are shown in pale cyan stick representation.

Figure 3.. Mechanism of ACAT-mediated cholesterol esterification.

(a) Overall structure of the ACAT1 tetramer (pdb: 6VUM). Cholesterol (yellow), acyl-CoA (grey), and nevanimibe (light pink) are shown as sticks. (b) Working model of ACAT enzymes, showing the catalytic histidine, cholesterol, and acyl-CoA.