CH 3 CH 2 OH +H 2 O CHO. 2e + 2H + + O 2 H 2 O +HCOOH

Transcription

1 2 4 H CH 3 2e + 2H H 2 2 H CH 2 H 2e + 2H H 2 2 H +H 2 CH 2e + 2H H 2 2 H +HCH Supplemental Figure S. The three-step 4DM reaction, each step requires two reducing equivalents from ADPH (transferred via the FAD and FM cofactors of cytochrome P450 reductase), two protons and one molecular oxygen. nly (H or CH 3 ) and 2 (H or =CH) vary in the 4DM substrates across the biological kingdoms. In the enzyme active center, the 4α-methyl group of the substrate is sequentially converted into the alcohol, then into the aldehyde derivative an then it is released as formic acid concomitantly with the introduction of the Δ 4-5 -double bound into the sterol core.

2 a i 47/280 =.8 b 448 nm 00 C/absolute, % Ligand free VI-bound Time (min) c 0. mm Supplemental Figure S2. Ligand-free (left/red) and VI-bound (right/blue) Tbb4DM. a. Absolute absorbance; b. C-difference spectra ( (Δt= min) Timecourse graph shows the rate and percentage of C-complexes formation in ligand-free and VI-bound state. c. Crystals.

3 G B B F K C B H G A A K I F F K L E J D J Supplemental Figure S3. A ribbon diagram showing the overall Tbb4DM structure. Distal view. -terminus (starting at G29) is colored in dark blue, and the C-terminus (ending at 476) is colored in red, full-length protein numbering. The 2 main helices are labeled from A to L; ten additional shorter helices between them are marked as ( ) and 2 β-strands arranged in 4 bundles are labeled by structure succession (5 strands in bundle, 2 in bundle 2, 3 in bundle 3, and 2 in bundle 4).The heme is shown in stick representation.

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5 Supplemental Figure S4. Sequence alignment of 4DMs from Trypanosomatidae (75% average amino acid sequence identity), Candida albicans (24% identity to Tbb4DM), human (27% identity) and Mycobacterium tuberculosis (27% identity). Secondary structure elements of 4DM from Tbb and Mt are shown at the top and the bottom, respectively. The P450-fold nomenclature is provided for Tbb4DM; differences in the secondary structure elements are indicated in red.

6 Supplemental Figure S5. Superimposition of four molecules of ligand-free (white) and four molecules of VI-bound (red) Tbb4DM (distal view).

7 G F I G H C K B D L K J J Supplemental Figure S6. View of the superposed ligand-free and VI-bound Tbb4DM structures from the proximal (opposite to Fig.2b) side. Some shift toward the heme in the VI-bound structure can be seen for helices C, D, H, J and the C-terminal part of helix G. Elongated secondary structure elements (helices C, H and G in all four molecules) are marked in red near the places of their extensions. The tendency to form additional helix-like turns has been also observed for azole-bound drug-metabolizing CYP2B4 [SU and 2V0M] and for substrate-bound CYP46A [2q9f] and might indicate some additional molecule surface stabilization.

8 a Y6 Y A29 F24 L V46 T295 I45 F48 b VI C422 Heme Supplemental Figure S7. 2Fo-Fc electron density map for VI in the active site of Tbb4DM contoured at σ (a) and 2.5 σ (b). The bond distances (Å) are marked.

9 a Substrate conversion (%) C. albicans 4DM 5min 60min Human 4DM b VI/4DM (molar ratio) Absorbance Kd=0.37 µm Wavelength, nm Supplemental Figure S8. a. Inhibition of C. albicans and human 4DM activity by VI. Enzyme concentration 0.5 µm, substrate (lanosterol) concentration 50 µm, SD<0%. The other details of the reaction conditions are as described in (9). b. Binding of VI to human 4DM, difference absorbance spectra. The human 4DM concentration.7 µm; mm VI solution in DMS, titration range µm, titration step 0.5 µm

10 a b c 00 B Y H V46 I/E 2 3 H T295 A29 Tbb C.alb Human 4 H I Supplemental Figure S9. VI amide group fragment as the most likely cause for its selectivity towards 4DM from pathogenic microbes. a. Comparison of inhibitory effects of VI (black bars) and clinical antifungal drug ketoconazole (white bars) on 4DMs from two pathogens, Tbb and Candida albicans, and from human. I/E 2 represents the inhibitor/enzyme ratio that causes a two-fold decrease in the initial rate of reaction, log scale. b. Chemical structures of ketoconazole (), VI (2) and two VI-scaffold derivatives: the β-phenyl azole SDZ (3) with the inhibitory potency and antiparasitic effect in Trypanosomatidae comparable to VI and the α-phenyl azole SDZ (4), a competitive, short-term inhibitor that binds with the same apparent Kd (<00 nm) but can be easily replaced in the enzyme active site by substrate (). c. VI-induced hydrogen bonding network between helices B and I. V46 and its mutation to isoleucine (grey), corresponding to I488 in human 4DM, are shown. Heme is presented as spheres.

11 Supplemental Table S. Data collection and refinement statistics Ligand-free Inhibitor-bound Data collection ative Iron SAD (peak) ative Wavelength, Å Space group P P P Cell dimensions a, b, c, Å 59.8,79.9, , 79.9, , 79., 6.0 α, β, γ, 74.2, 8.6, , 8., , 79., 68.6 umber of molecules per asymmetric unit esolution, Å ( )* ( ) (.9-.87) merge (0.470) (0.536) (0.603) I/σ(I) 8.5 (3.) 44.5 (3.4) 28 (.8) Completeness (%) 97.8 (96.7) 96.8 (95.2) (9.7) edundancy 4.0 (3.8) 7.8 (7.7) 3.9 (3.5) efinement esolution, Å umber of reflections 42,726 44,527 work/ / free 0.95/ /0.238 Average B-factors protein heme/inhibitor 22 23/32 water ms deviations Bond lengths, Å Bond angles, Cα positions between 4 molecules, Å *Values in parenthesis are for highest-resolution shell.