Henlea earthworm bioluminescence comprises violet-blue BRET from tryptophan 2-carboxylate to deazaflavin cofactor

• Published in: Biochemical and biophysical research communications 2024-05, Vol.708, p.149787, Article 149787
• Main Authors: Petushkov, Valentin N., Vavilov, Matvey V., Khokhlova, Anastasia N., Zagitova, Renata I., Belozerova, Olga A., Shcheglov, Aleksandr S., Kovalchuk, Sergey I., Tsarkova, Aleksandra S., Rodionova, Natalia S., Yampolsky, Ilia V., Dubinnyi, Maxim A.
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - metabolism; Biological Products; Bioluminescence; Fluorescence; Henlea; Luminescent Proteins - metabolism; Mass-spectrometry; Oligochaeta - metabolism; Total synthesis; Tryptophan
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2024.149787

We recently identified the deazaflavin cofactor as a light emitter in novel bioluminescence (BL) system from Siberian earthworms Henlea sp. (Petushkov et al., 2023, Org. Biomol. Chem. 21:415–427). In the present communication we compared in vitro BL spectra in the absence and in the presence of the cofactor and found a wavelength shift from 420 to 476 nm. This violet-blue BRET to deazaflavin cofactor (acceptor of photonless transfer) masks the actual oxyluciferin as an emitter (BRET donor) in the novel BL system. The best candidate for that masked chromophore is tryptophan 2-carboxylate (T2C) found previously as a building block in some natural products isolated from Henlea sp. (Dubinnyi et al., 2020, ChemSelect 5:13155–13159). We synthesized T2C and acetyl-T2C, verified their presence in earthworms by nanoflow-HRMS, explored spectral properties of excitation and emission spectra and found a chain of excitation/emission maxima with a perfect potential for BRET: 300 nm (excitation of T2C) - 420 nm (emission of T2C) - 420 nm (excitation of deazaflavin) - 476 nm (emission of deazaflavin, BL). An array of natural products with T2C chromophore are present in BL earthworms as candidates for novel oxyluciferin. We demonstrated for the Henlea BL that the energy of the excited state of the T2C chromophore is transferred by the Förster mechanism and then emitted by deazaflavin (BRET), similarly to known examples: aequorin-GFP in Aequorea victoria and antenna proteins in bacterial BL systems (lumazine from Photobacterium and yellow fluorescent protein from Vibrio fischeri strain Y1). •Tryptophan 2-carboxylates are widespread compounds in bioluminescent Henlea earthworms.•The tryptophan 2-carboxylate was synthesized and unambiguously characterized by NMR, MS and UV for the first time.•The hypothesis of tryptophan 2-carboxylate-to-deazaflavin FRET is formulated for novel bioluminescent system.•Bioluminescence spectra with and without deazaflavin were measured and confirmed the violet-blue BRET in Henlea.•The BRET in nature was previously reported only as aequorin-GFP transfer and for antenna proteins in bacterial BL systems.