Abstract
We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-A structure of ligand-free CYP125 reveals a "letterbox" active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val(267) residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val(267), suggesting a substrate binding-induced low- to high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.
Highlights
We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice
Econazole is effective in clearing Mycobacterium tuberculosis (Mtb) infection in a mouse model, and recent studies on Mtb CYP130 revealed the binding mode of the drug to this P450 (16, 18)
Solvent treatments of HS CYP125 fractions did not result in extraction of potential substrates bound to the enzyme, but did demonstrate that the heme spin state could be readily modulated by organic solvents
Summary
CYPI25 Cloning, Expression, and Purification—CYP125 was cloned by PCR from a Mtb H37Rv cosmid library (from Institut Pasteur, Paris). Induced optical change versus ligand concentration data were fitted using Equation 1, which provides the most accurate estimation of Kd values for the tight binding azole drugs, as we have described in previous studies of the Mtb CYP121 and CYP51B1 P450s (8, 17). Absorption change versus applied potential data were fitted to the Nernst function (using Origin software) to derive the midpoint potential for the CYP125 heme iron Fe3ϩ/Fe2ϩ couple (29). Energy minimization (1000 steps, conjugate gradient) and MD simulations (200 ps) were initially performed in vacuo at 100 K to thermally equilibrate CYP125-cholesterol complexes. Final minimization (1000 steps, conjugate gradient) was performed to obtain the CYP125-cholesterol complexes. Comparison and selection of the docked cholesterol models was done by comparing the stabilization energy due to the CYP125-cholesterol interactions (supplemental Table S4) and the minimal distances between cholesterol heavy atoms and the iron atom of the heme. All other reagents were from Sigma and were of the highest grade available
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