Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs

Abstract

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.

Keywords: Aspergillus; antifungal drugs; cytochrome P450; drug design; enzyme inhibitor; sterol; sterol 14alpha-demethylase (CYP51); x-ray crystallography.

FIGURE 1.

CYP51 inhibitors and catalytic reaction. A, structural formulas of the marketed drugs, 1,2,4-triazoles voriconazole (Vor), fluconazole (Fluc), and posaconazole (Poz), and an experimental CYP51 inhibitor, the 1,3-imidazole VNI. The heme-coordinating nitrogen atoms are circled. B, 14α-demethylation of eburicol. The reaction involves three consecutive P450 catalytic cycles, each consuming two electrons (provided by CPR, plus two protons) to reduce the catalytic heme iron and activate the molecular oxygen, resulting in the insertion of one oxygen atom into the substrate and reduction of the other oxygen atom to water. In this reaction, the sterol 14α-methyl group is first converted into the 14α-alcohol and then into the 14α-aldehyde (CHO-) intermediate and finally released as formic acid. C, HPLC profile of sterols extracted after a 5-min reaction of A. fumigatus CYP51B with eburicol (37 °C; P450, CPR, and eburicol concentrations were 0.5, 1, and 25 μm, respectively).

FIGURE 2.

Sequence alignment of CYP51 proteins from A. fumigatus (A.fuB and A.fuA), human, and a protozoan pathogen, T. brucei (T.bru). The alignment was generated in ClustalW and processed in ESPript to add secondary structure information on A.fuB (top) and T.bru (bottom), using molecules A of Protein Data Bank files 4UYL and 3GW9, respectively. The amino acid sequence identity between A.fuB and A.fuA is 59%. Within the Aspergillus genus, CYP51B identities range from 78% (Aspergillus niger) to 99% (Aspergillus fischerianus), whereas CYP51A identities range from 69% (Aspergillus nidulans) to 96% (A. fischerianus) (not shown). The identities between A.fuB versus human and A.fuB versus T.bru CYP51 amino acids are 33 and 23%, respectively. The alignment shows that, regardless of low amino acid sequence identity, the length and location of the secondary structural elements in A.fuB and T.bru CYP51s match very well, except for the FG arm segment, which is longer in T.bru (in green), and the additional β-bundle in A.fuB (strands β5-1 and β5-2, in blue); this segment appears to be specific to fungal CYP51 and so far has not been seen in the structures of CYPs from any other families. The CYP51-specific helical turn in the SRS1 region (ηB″) is shown in brown. The heme-coordinated cysteine and the conserved CYP51-specific histidine (proton delivery, helix I) are colored in yellow; the five residues bonded with porphyrin propionates are marked with asterisks.

FIGURE 3.

Spectral characteristics of A. fumigatus CYP51B. A, ligand-free full-length protein. Shown are the absolute absorbance spectrum of the ferric (Fe³⁺) state (Soret band maximum at 418 nm) and the difference absorbance spectrum of the reduced CO-bound state (Soret band maximum at 448 nm). The P450 concentration was 3.3 μm, and the ratio (ΔA₃₉₃ − A₄₇0)/ΔA₄₁₈ − A₄₇₀) was 0.40. B, truncated protein co-purified with VNI and used for crystallization. Shown is the absolute absorbance spectrum; spectrophotometric index A₄₂₅/A₂₈₀ = 1.3. The P450 concentration was 2.8 μm. Inset, 12% (w/v) SDS-PAGE electrophoretogram. Left lane, rainbow marker; middle and right lanes, P450 after Ni²⁺-NTA-agarose and CM-Sepharose chromatography, respectively (54,000 Da).

FIGURE 4.

Time course of enzymatic reduction (detected as the CO complex) of A. fumigatus CYP51B in the presence and in the absence of substrate (1.5 μm P450, 3.0 μm CPR, 150 μm NADPH). Optical path length was 1 cm. Insets, difference CO-binding spectra, Δt = 2 min.

FIGURE 5.

Spectral responses of A. fumigatus CYP51B to the addition of sterols. A, eburicol. B, lanosterol. C, obtusifoliol. Absolute (top) and difference (bottom) absorbance spectra are shown. S, sample; R, reference. P450 concentration was 5 μm, and optical path length was 1 cm. The titration curves (obtained using Equation 1) are shown in the insets, and the K_d values are corrected for cyclodextrin binding. The C24-methylene group appears to be required for the proper binding of sterol substrates to A. fumigatus CYP51B.

FIGURE 6.

Spectral response of A. fumigatus CYP51B to the addition of the heme-coordinating ligands VNI and voriconazole. Absolute (top) and difference (bottom) absorbance spectra are shown. The P450 concentration was 1.0 μm, and the optical path length was 5 cm. The titration curves (obtained using Equation 1) are shown in the inset.

FIGURE 7.

Enzymatic activity of A. fumigatus CYP51B. A, time course of substrate conversion at 37 °C (0.5 μm P450, 1.0 μm CPR, and 25 μm eburicol), shown in comparison with C. albicans CYP51. B, steady-state kinetics of A. fumigatus CYP51B with its natural substrate eburicol and obtusifoliol (a substrate for plant CYP51 enzymes). The P450 concentration was 0.5 μm (37 °C, 60-s reaction). The experiments were performed in triplicate, and results are presented as means ± S.E. (error bars).

FIGURE 8.

Inhibitory effects of azoles on the activity of A. fumigatus CYP51B. The incubation time was 60 min (at 28 °C). The molar enzyme/inhibitor/substrate ratio was 1:2:50, with 0.5 μm P450. The experiments were performed in triplicate, and results are presented as means ± S.E. (error bars).

FIGURE 9.

Overall view of the A. fumigatus CYP51 structures. A, view along the rotation axis that runs from top to bottom and relates two VNI (pink)-A. fumigatus CYP51B complexes that comprise the asymmetric unit. The secondary structural elements forming the mouth of the substrate access channel (helices A′ and F″ and the β4-hairpin) are marked. B, superimposition of four molecules of A. fumigatus CYP51B. Shown is a distal P450 view; x/y/z: 65/67/45 Å. Molecule A of the VNI complex is shown in a ribbon representation; the backbones of the other three molecules are presented as wires. VNI and voriconazole are deleted for clarity. In both panels, the protein backbone is presented in rainbow coloring from blue (N terminus) to red (C terminus). The heme is shown as a stick model, and the iron is depicted as an orange sphere. C, stereo view of B.

FIGURE 10.

Family-specific structural features of A. fumigatus CYP51B. A, the active site cavity. Eburicol (green) was modeled in a position similar to the substrate analog MCP as described under “Experimental Procedures.” The protein backbone is colored by secondary structure (helices are red, β-strands are blue, loops are gray, and turns are green). The corresponding SRS1 area in T. brucei CYP51 is shown with a light yellow ribbon. The B′ helical turn, helix C, and the β5-bundle are marked. B, heme support from the protein moiety. Hydrogen bonds are shown as green dashes. C, proton transfer route. His³¹⁰ is conserved across the CYP51 family and in all known CYP51 structures forms a hydrogen bond with an acidic residue (aspartate or glutamate) of the F helix. The length of the His³¹⁰-Asp²²⁷ hydrogen bond in the four A. fumigatus CYP51B molecules is 2.8 ± 0.1 Å (mean ± S.E.). Ser³¹¹ corresponds to the conserved P450 threonine.

FIGURE 11.

Voriconazole binding mode. A, view of the A. fumigatus CYP51B active site illustrating interactions of the hemoprotein with the inhibitor. The residues located within van der Waals contacts (<4.5 Å) with voriconazole are depicted as wire models and labeled; the carbon atoms are colored in gray; and three reference secondary structural elements are shown as semitransparent blue ribbon. The carbon atoms of voriconazole and the heme (stick representations) are blue and orange, respectively. The hydrogen bonds are shown as green dashed lines. B, 2F_o − F_c omit electron density map of the active site area around voriconazole contoured at 1.5σ. C, superimposition of voriconazole complexes with A. fumigatus CYP51B and with CYP46 (Protein Data Bank code 3MDT). The heme and the drug are shown in a stick representation; the carbon atoms are blue and gray in A. fumigatus CYP51B and CYP46, respectively.

FIGURE 12.

VNI binding mode. A, 2F_o − F_c omit electron density map of the active site area around VNI contoured at 1.3 σ. B, superimposition of the VNI complexes with A. fumigatus CYP51B and T. brucei CYP51 (Protein Data Bank code 3GW9). The heme and VNI are shown in a stick representation; the carbon atoms are magenta and gray in the A. fumigatus and T. brucei enzymes, respectively. The hydrogen bonds are presented as green dashed lines. Two segments of the T. brucei CYP51 molecule that are connected by the hydrogen bond network with VNI (helices B′ and I) are outlined as gray ribbons. C, view of the A. fumigatus CYP51B active site illustrating its interactions with VNI. The residues located within van der Waals distances (<4.5 Å) with VNI are depicted as wire models and labeled, and the carbon atoms are colored in gray. Three reference secondary structural elements are seen as a semitransparent pink ribbon. The carbon atoms of VNI and the heme (stick representations) are colored magenta and orange, respectively.

FIGURE 13.

VNI-induced rearrangements in A. fumigatus CYP51B (semitransparent pink structure, except for Phe²³⁴ and VNI, which are colored in magenta). Superimposition with the voriconazole co-structure (semitransparent blue) is shown. The π-π stacking interactions between VNI and Phe²³⁴ are depicted as black dashed lines; the distances are marked. The hydrogen bonds are green. Helices A′ and F″ and the β4-hairpin are the elements forming the entrance into the CYP51 substrate access channel. The heme is seen as a gray sphere model. The other 4 residues in the superimposed complex with voriconazole (Leu⁹², Met²³⁵, His³¹⁰, and Phe⁵⁰⁴), whose side chain locations differ substantially, are shown as semitransparent blue lines; the directions of the rearrangements are indicated with gray arrows.

FIGURE 14.

Superimposed structures of A. fumigatus (4UYL, pink), T. brucei (3GW9, blue), and human (3LD6, yellow) CYP51 enzymes. A, distal view. The protein backbone is presented as a semitransparent ribbon, and the area of the substrate-binding cavity is within the black square. B, enlarged view of the binding cavity. The heme and the residues invariant in all known CYP51 enzymes (>300 sequences) are depicted in a stick representation with T. brucei CYP51 numbering (see also Fig. 2).

FIGURE 15.

Substrate entry. A, superimposition of A. fumigatus (4UYL, colored according to secondary structure as in Fig. 10) with T. brucei CYP51 (3SW9, gray; the FG-arm is yellow). The distal view is shown. The length of the FG arm in A. fumigatus CYP51B is shorter (by ∼5 Å) than it is in the T. brucei enzyme. B, surface representation of CYP51s from different phyla. 1, voriconazole-bound A. fumigatus CYP51; 2, VNI-bound A. fumigatus CYP51; 3, ketoconazole-bound human CYP51; 4, MCP-bound T. brucei CYP51. The ligands are shown as spheres with green carbon atoms. There is no opening in the upper surface of the human or protozoan CYP51 structure.

FIGURE 16.

Fungi-specific postmeander insert (β5-bundle). A, proximal view of the CYP51 molecule, colored by secondary structure as in Fig. 10. Insets, electrostatic potential mapped onto the proximal surface. Red, negative charge; blue, positive charge. The β5-area is circled. B, a fragment of multiple CYP51 sequence alignment.

FIGURE 17.

A. fumigatus CYP51B and CYP51A may differ in their substrate preferences. A, substrate binding cavities in A. fumigatus CYP51B and T. brucei CYP51. A. fumigatus CYP51B has more space in the area holding the distal portion of the sterol aliphatic arm (circled). B, a fragment of multiple sequence alignment of CYP51s A and B from filamentous fungi. A.fum, A. fumigatus; A.flav, Aspergillus flavus; A.lent, Aspergillus lentulus; A.clav, Aspergillus clavatus; N.fisch, Neosartorya fischeri; T.tons, Trichophyton tonsurans; F.gram, Fusarium graminearum; V.inaec, Venturia inaequalis; U.necator, Uncinula necator; C.neoform, Cryptococcus neoformans; U.maydis, Ustilago maydis. C, location of Thr²⁸⁵ (yellow), which aligns with Ala³⁰³ in A. fumigatus CYP51B (blue) in the molecular model of A. fumigatus CYP51A.