SignificanceWe present identification of the luciferase and enzymes of the biosynthesis of a eukaryotic luciferin from fungi. Fungi possess a simple bioluminescent system, with luciferin being only two enzymatic steps from well-known metabolic pathways. The expression of genes from the fungal bioluminescent pathway is not toxic to eukaryotic cells, and the luciferase can be easily co-opted to bioimaging applications. With the fungal system being a genetically encodable bioluminescent system from eukaryotes, it is now possible to create artificially bioluminescent eukaryotes by expression of three genes. The fungal bioluminescent system represents an example of molecular evolution of a complex ecological trait and with molecular details reported in the paper, will allow additional research into ecological significance of fungal bioluminescence.

Study of fungal bioluminescence mechanisms generates development of a multicolor enzymatic chemiluminescence system.

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30. All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands. The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458. Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect. Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand. The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE. These methods also confirmed the absence of interaction of the holoflavoproteins.

The solvation dynamics of interesting bioluminescent chromophores have been determined, using subpicosecond and wavelength-resolved fluorescence spectroscopy, in combination with global analysis of the multidimensional data sets. The systems investigated comprise the free ligands 6,7-dimethyl-(8-ribityl)-lumazine (lumazine) and riboflavin in an aqueous buffer and both ligands when noncovalently bound to two bacterial bioluminescent antenna proteins: lumazine protein (from Photobacterium leiognathi) and the blue fluorescent protein (from Vibrio fischeri Y1). Fluorescence spectral relaxation of the free ligands is complete within a few picoseconds. Subsequently, the fluorescence intensity increases by ∼7% on a time scale of 15-30 ps. Fluorescence spectral relaxation of the protein-bound ligands is largely complete within 1 ps but reveals a small red shift with a minor, but distinctly longer, relaxation time than that of the free ligands, which is tentatively assigned to the relaxation of protein-bound water in the vicinity of the excited chromophore.

Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system’s low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.

We report structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota. This luciferin represents a key component of a novel ATP-dependent bioluminescence system. The UV, fluorescence, NMR and HRMS spectral studies were performed on 5 mkg of the isolated substance, and gave four isomeric structures, conforming with spectral data. These isomers were chemically synthesized and one of them was found to produce light in the reaction with a protein extract from Fridericia. The novel luciferin was found to have an unusual deeply modified peptidic nature, implying an unprecedented mechanism of action.

Time-resolved fluorescence was used to directly measure the energy transfer rate constant in the protein-protein complex involved in the yellow bioluminescence of Vibrio fischeri, strain Y1. In this reaction the putative donor is the fluorescent transient intermediate, luciferase hydroxyflavin, which exhibits a major fluorescence lifetime of the bound flavin of 10 ns. On addition of the acceptor, the V. fischeri yellow fluorescence protein containing either FMN or riboflavin as ligand, a rapid decay time, 0.25 ns, becomes predominant. The same results are observed using rec-luciferase from Photobacterium leiognathi to produce the donor. Because of favorable spectral separation in this system, this rapid decay rate of 4 ns-1, can be directly equated to the energy transfer rate. This rate is ten times higher than the rate previously observed in the Photobacterium luciferase hydroxyflavin-lumazine protein, donor-acceptor system, derived from emission anisotropy measurements. This ten-times ratio is close to the ratio of spectral overlaps of the donor fluorescence with the acceptor absorption, between these two systems, so it is concluded that the topology of the protein complexes in both cases, must be very similar. Energy transfer is also monitored by the loss of steady-state fluorescence intensity at 460 nm of the donor, on addition of the acceptor protein. A fluorescence titration indicates that luciferase hydroxyflavin and the yellow protein complex with a 1:1 stoichiometry with a Kd of 0.7 microM (0 degree C). These parameters account for the bioluminescence spectral shifting effects observed in these reactions.