Structural Basis for P450-Mediated Oxysterol Metabolism by Mycobacterium Tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. Mtb can avoid immune response, however the mechanisms are not fully investigated. Recently we have shown that Mtb cholesterol degrading enzymes, CYP124, CYP125, CYP142 and 3βHSD, metabolize a wide variety of human immune oxysterol messengers. The list includes: a) 25-hydroxy-cholesterol (agonist of LXR); b) 7β-hydroxy-cholesterol (agonist of orphan RORα/γ); c) 7α,25-dihydroxy-cholesterol (agonist of the EBI2); d) 20S-hydroxy-cholesterol (a ligand of Smoothened in the Hedgehog signaling pathway). CYP124 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives, which induces transcription of antimicrobial peptides - cathelicidin and β-defensin 2, through VDR. Here we give a structural basis for binding and oxidation of a wide variety of substrates by CYP124. Atomic resolution structures of CYP124, complexed with cholestenone (1.65 Å resolution), VD3 (1.3 Å) and 1α-hydroxy-VD3 (1.2 Å) reveal a site for oxidative attack during catalysis. In all the structures, binding pocket tightly locks aliphatic tails, leaving more degrees of freedom for positioning of the sterols A-rings. Moreover, SQ109 - a prospective antitubercular compound, which is structurally resembles the oxysterols and secosteroids, was also metabolized by CYP124, and we have solved 1.25 Å-resolution structure of the complex as well. Finally, we have also obtained high resolution crystal structures of CYP124, complex with two micromolar inhibitors, contributing to the drug design, based on the suppression of immune signal molecules degradation. This work was supported by a joint research grant of the Belarusian Republican Foundation for Fundamental Research (B20R-061) and the Russian Foundation for Basic Research (20-54-00005). VB and AM are supported by the Ministry of Science and Higher Education of the Russian Federation (agreement # 075-00337-20-03, project FSMG −2020-0003).