Ligand-binding properties and catalytic activity of the purified human 24-hydroxycholesterol 7α-hydroxylase, CYP39A1

•Human CYP39A1 was purified to homogeneity and characterized at the protein level for the first time.•The CYP39A1 binds oxysterols and sex steroids with high affinity. However, metabolism was detected only with 24S-hydroxycholesterol.•CYP39A1 function can be regulated by azole and steroid drugs. Oxysterols are derivatives of cholesterol and biologically active molecules that are involved in a number of functions, including cholesterol homeostasis, immune response, embryogenic development and pathophysiology of neurodegenerative diseases. Enzymes catalyzing their synthesis and metabolism are of particular interest as potential or evaluated drug targets. Here we report for the first time biochemical analysis of purified human oxysterol 7α-hydroxylase selective for 24-hydroxycholesterol. Binding analyses indicated a tight binding of the oxysterols and estrone. Ligand screening revealed that CYP39A1 binds with high affinity antifungal drugs and prostate cancer drug galeterone (TOK-001). Site-directed mutagenesis of conserved Asn residue in the active site revealed its crucial role for protein folding and heme incorporation. Developed protocol for expression and purification enables further investigation of this hepatic enzyme as off-target in development of specific drugs targeting cytochrome P450 enzymes.