X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51)

Abstract

A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.

FIGURE 1.

DHBP binding to CYP51_Mt. The dissociation constant, K_D, of DHBP binding was estimated from the absorbance shift at 420 nm during spectroscopic titration of CYP51_Mt with increasing concentrations of DHBP. A fragment of the series of difference spectra is shown in the inset.

FIGURE 2.

Overall structure of the CYP51_Mt-DHBP complex. Stereo of the superimposed CYP51_Mt forms bound to DHBP (green) or to 4-phenylimidazole (gray) (PDB ID code 1E9X) is shown. The arrows indicate the directions of the movement for the most mobile structural elements upon binding DHBP. Relocation distances are shown in Angstroms. The helices C, G, and H are represented by tubes. Heme is in orange, and DHBP in cyan. Images here and in Fig. 4 are generated using the VMD software (76).

FIGURE 3.

DHBP binding in the CYP51_Mt active site. A stereo view of DHBP bound in the active site of CYP51_Mt is shown. C atoms are colored in cyan (DHBP) or green (protein), N atoms are shown in blue, O atoms are shown in red, and S atoms are shown in yellow. Amino acid side chains within 4 Å from DHBP are labeled in black, and Phe-89 (within 7.3 Å) is labeled in blue. Fragments of the 2F_o - F_c electron density composite omit map contoured at 1.0 σ (pale cyan and cyan) and 0.6 σ (red) are shown as mesh. To avoid excessive cluttering, heme (orange) was excluded from the map calculation. The image was generated using PyMOL (77).

FIGURE 4.

DHBP versus sterol binding in CYP51_Mt active site. A stereo view of DHBP (pale cyan) surrounded by the amino acid side chains (cyan) within 6 Å plus Phe-89 (within 7.3 Å) is shown. DHBP is superimposed with estriol (from the experimental structure with the PDB ID code 1X8V) (A) or lanosterol (as modeled elsewhere (37) by molecular dynamic simulations) (B). Sterols in A and B are shown as light gray shadows. Heme is in orange.

SCHEME 1.

Substrates (A) and type I binding ligands (B) of CYP51Mt.

FIGURE 5.

Inhibitory effects of DHBP (A) and EPBA (B) in mouse macrophages. The reduction in the bacterial colony-forming unit (CFU) count at 1–5 days after macrophage infection with M. tuberculosis H37Rv as a result of macrophage treatment with increasing concentrations of DHBP (A): (▪) non-treated, (▴) 50 μm DHBP, (▾) 100 μm DHBP or EPBA (B): (▪) non-treated, (×)10 μm EPBA, (○) 30 μm EPBA is shown.