This project continues the RSF grant 21-14-00382 (2021-2023, granted to Dubinny M.A.) “Structure and metabolism of luciferin in bioluminescent enchytraeids Henlea sp.”, submitted from the same organization, has the same team and the same leader. The application was submitted by a separate project in connection with an official letter from the Russian Science Foundation (dated October 27, 2023) about the cancellation this year of the competitive selection for the extension of the terms of projects of individual scientific groups previously supported by RSF grants (extension of 2021 projects).

Expansion of the list of bioluminescent tools with a new unique BL system of the enchytraeid Henlea sp. is the key goal of this project. This application sets an ambitious goal: to complete the study of the fundamentally new bioluminescent system of Henlea sp. to a wide range of practical applications. During our previous RSF project, the bioluminescent reaction emitter of Henlea sp. was discovered, identified, and chemically synthesized. It was a new unusual ActH cofactor based on deazaflavin, which reacts with bioluminescence with a micromolar Michaelis constant and enhances bioluminescence by 33 times (Petushkov et al. 2022). We have established the chemical structure of luciferin from Henlea sp., which turned out to be a new, previously unknown tryptophan-based compound. Using nanoflow-MS/MS spectroscopy, the oxidation of natural luciferin was carried out and one of the products of the bioluminescent reaction, tryptophan-C2 carboxylate, was unambiguously identified. The Henlea sp. transcriptome was sequenced, a proteomic analysis of a crudely purified luciferase preparation was performed, and candidates for the role of the gene encoding Henlea sp. luciferase were identified.

While working on the project we plan:

1. Chemically synthesize the putative oxidation products of luciferin - tryptophan-C2 carboxylate esters and, by comparison with the oxidation product of natural luciferin, establish the exact chemical structure of the true oxyluciferin of Henlea sp., the spontaneous breakdown of which leads to the appearance of tryptophan-C2 carboxylate;

2. Synthesize two stereoisomers of Henlea sp luciferin (β and γ-luciferins). and carry out an enzymatic bioluminescent oxidation reaction;

3. Synthesize (at least one) Henlea sp luciferin analogue to identify functionally important luciferin groups;

4. Unambiguously identify the luciferase gene and express it as active luciferase.

The result of the work on the RSF project will be a new bioluminescent system of the Siberian enchytraeids Henlea sp. ready for practical use outside our narrow scientific group.