Research ArticleBIOCHEMISTRY

Mechanism and color modulation of fungal bioluminescence

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Science Advances  26 Apr 2017:
Vol. 3, no. 4, e1602847
DOI: 10.1126/sciadv.1602847
  • Fig. 1 Characterization of the fungal oxyluciferin.

    (A) General mechanism of the fungal bioluminescence and synthesis of the oxyluciferin (2). Hispidin is hydroxylated by a styrylpyrone hydroxylase [hispidin-3-hydroxylase (H3H)] in the presence of O2 and NAD(P)H, producing 3-hydroxyhispidin (1) (11), the fungal luciferin, which is enzymatically oxidized by O2, giving an HEI that decomposes in CO2 and the excited oxyluciferin. Fluorescence emission gives the ground-state oxyluciferin (2). The oxyluciferin was synthesized in two steps from 3,4-dimethoxybenzalacetone. LiHMDS, lithium bis(trimethylsilyl)amide; THF, tetrahydrofuran. (B) Comparison of HPLC-PDA-ESI-MS profiles of the enzymatic reaction (after 15 min) and the synthetic oxyluciferin. mAU, milliarbitrary unit. (C) Mass spectra of the luciferin 1 (Rt = 10.1 min, m/z = 261 [M − H]) and compound 2 (Rt = 12.2 min, m/z = 249 [M − H]). (D) Matching of the fungal bioluminescence (BL) spectrum and the fluorescence (FL) spectrum of 2 in acetone. The absorption spectrum of 2 is shown for reference (λmax = 380 nm).

  • Fig. 2 Experimental and theoretical study of the oxidation of 3-hydroxyhispidin.

    (A) Cyclic voltammetry of hispidin and the luciferin in aqueous KCl. (B) Spin density surface of the radical cations of hispidin and 3-hydroxyhispidin and the corresponding deprotonated radicals.

  • Fig. 3 Formation of the HEI via the oxidation of 3-hydroxyhispidin.

    (A) General mechanistic proposal for the enzymatic generation of the HEI and the chemical preparation of the endoperoxide of 3 via methylene blue–sensitized photo-oxygenation with [18O]-labeled singlet oxygen [1(18O2)]. (B) Difference between the normalized mass spectra signal intensity before and after photo-oxygenation of 3 and the structure proposal for product ion at an m/z of 163. Molecular structures represent the neutral form of 1 and 3, not the anion detected by MS.

  • Fig. 4 Color modulation of fungal bioluminescence.

    (A) Natural luciferin (1), synthetic analogs 3 and 5 to 9, and enzymatic chemiluminescent reaction in tris buffer (pH 7). (B) Chemiluminescence spectra of luciferins 1, 3, and 6 to 9. The Savitzky-Golay filter (20 points) was used to improve the signal-to-noise ratio due to very low light intensity. (C) Chemiluminescence quantum yields (ΦCL) and (D) the observed chemiluminescence decay rate constant for compounds 1, 3, and 6 to 9.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1602847/DC1

    Synthetic procedures

    fig. S1. Determination of fluorescence quantum yield of the oxyluciferin.

    fig. S2. Determination of the chemiluminescence and singlet quantum yields of the luciferin/luciferase reaction.

    fig. S3. Dependence of the chemiluminescence intensity from luciferin/luciferase reaction on the concentration of luciferin (76 to 760 nM) and phosphate buffer (pH 6 to 8).

    fig. S4. Photocathode spectral response curve provided by Berthold Detection Systems (PMT type 9107).

    fig. S5. Dynamics of the reaction between substrate and luciferase microsomal preparation of N. nambi.

    fig. S6. Chemiluminescence spectra of 1 (orange curve) and 3 (green curve) and the fluorescence spectrum of 4 in acetone (dotted curve).

    fig. S7. Chemiluminescence from the reaction of 1,4-dimethylnaphthalene endoperoxide (DMNO2) with 3-hydroxyhispidin (luciferin).

    fig. S8. Collision-induced dissociation spectrum (ESI-MS/MS) of the labeled oxyluciferin—compound 4.

    fig. S9. Absorption (blue) and fluorescence emission (red) spectra of compounds 2, 3, and 5 to 9.

    fig. S10. Isolation and identification of compounds in peaks 2 and 4 of the luciferin 1/luciferase reaction.

    fig. S11. Proposed mechanisms for oxyluciferin degradation.

    fig. S12. Synthesis of the fungal luciferin 1.

    fig. S13. Synthesis of 3,4-dihydroxy-6-methyl-2H-pyran-2-one 13.

    fig. S14. Synthesis of compounds 14 to 16.

    fig. S15. Synthesis of fungal luciferin analogs 3 and 5 to 9.

    fig. S16. Synthesis of fungal oxyluciferins 2 and 4.

    fig. S17. NMR spectra chemical shifts of compounds 18 to 23.

    fig. S18. NMR spectra chemical shifts of luciferin analogs 3 and 5 to 9.

    table S1. Observed decay rate constants (kobs), the total light emitted (Q) by the reaction of luciferin/luciferase reaction, and chemiluminescence and singlet quantum yields (ΦCL and ΦS) at different pH and substrate concentrations.

    table S2. Dependence of chemiluminescence quantum yield (ΦCL) and singlet quantum yield (ΦS) on the pH of luciferin-luciferase reaction.

    table S3. Spectral characteristics of compounds 1 to 3 and 5 to 9.

    table S4. Observed decay rate constant (kobs), the total of light emission (Q), photomultiplier sensitivity correction factor (fPMT), and the chemiluminescence quantum yield (ΦCL) obtained from the reaction between the crude extract of N. gardneri and compounds 1, 3, and 5 to 9.

  • Supplementary Materials

    This PDF file includes:

    • Synthetic procedures
    • fig. S1. Determination of fluorescence quantum yield of the oxyluciferin.
    • fig. S2. Determination of the chemiluminescence and singlet quantum yields of the luciferin/luciferase reaction.
    • fig. S3. Dependence of the chemiluminescence intensity from luciferin/luciferase reaction on the concentration of luciferin (76 to 760 nM) and phosphate buffer (pH 6 to 8).
    • fig. S4. Photocathode spectral response curve provided by Berthold Detection Systems (PMT type 9107).
    • fig. S5. Dynamics of the reaction between substrate and luciferase microsomal preparation of N. nambi.
    • fig. S6. Chemiluminescence spectra of 1 (orange curve) and 3 (green curve) and the fluorescence spectrum of 4 in acetone (dotted curve).
    • fig. S7. Chemiluminescence from the reaction of 1,4-dimethylnaphthalene endoperoxide (DMNO2) with 3-hydroxyhispidin (luciferin).
    • fig. S8. Collision-induced dissociation spectrum (ESI-MS/MS) of the labeled oxyluciferin—compound 4.
    • fig. S9. Absorption (blue) and fluorescence emission (red) spectra of compounds 2, 3, and 5 to 9.
    • fig. S10. Isolation and identification of compounds in peaks 2 and 4 of the luciferin 1/luciferase reaction.
    • fig. S11. Proposed mechanisms for oxyluciferin degradation.
    • fig. S12. Synthesis of the fungal luciferin 1.
    • fig. S13. Synthesis of 3,4-dihydroxy-6-methyl-2H-pyran-2-one 13.
    • fig. S14. Synthesis of compounds 14 to 16.
    • fig. S15. Synthesis of fungal luciferin analogs 3 and 5 to 9.
    • fig. S16. Synthesis of fungal oxyluciferins 2 and 4.
    • fig. S17. NMR spectra chemical shifts of compounds 18 to 23.
    • fig. S18. NMR spectra chemical shifts of luciferin analogs 3 and 5 to 9.
    • table S1. Observed decay rate constants (kobs), the total light emitted (Q) by the reaction of luciferin/luciferase reaction, and chemiluminescence and singlet quantum yields (ΦCL and ΦS) at different pH and substrate concentrations.
    • table S2. Dependence of chemiluminescence quantum yield (ΦCL) and singlet quantum yield (ΦS) on the pH of luciferin-luciferase reaction.
    • table S3. Spectral characteristics of compounds 1 to 3 and 5 to 9.
    • table S4. Observed decay rate constant (kobs), the total of light emission (Q), photomultiplier sensitivity correction factor (fPMT), and the chemiluminescence quantum yield (ΦCL) obtained from the reaction between the crude extract of N. gardneri and compounds 1, 3, and 5 to 9.

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