Melatonin Is Bound to a Glycolytic Protein Complex via Tdh2p During Fermentation in Saccharomyces cerevisiae

Melatonin (MEL) is a multifunctional bioactive compound known for its antioxidant properties and regulatory roles in both animal and microbial systems. Although MEL has been shown to interact with glycolytic enzymes in yeast, the molecular basis and physiological relevance of these interactions remain unclear. Here, we investigate MEL interactions with key glycolytic proteins in Saccharomyces cerevisiae during alcoholic fermentations, using CRISPR-Cas9-generated gene deletion mutants. Protein interactions in wild type and mutant strains were analysed via co-immunoprecipitation and western blotting. The fermentative behavior of single and double TDH deletion mutants was assessed in both laboratory and wine yeast strains. TDH2 and TDH3 deletions extended the lag phase and reduced GAPDH activity, while TDH1 deletion had minor effects, indicating a differential contribution of glycolytic isoenzymes to fermentation kinetics. Analysis of MEL-bound proteins revealed that Tdh2p is essential for complex formation, as its absence abolished MEL binding to other glycolytic proteins. Molecular docking predicted stable hydrogen bonds between MEL and Tdh2p, supporting a direct interaction. These results demonstrate the formation of a glycolytic protein-MEL complex and identify Tdh2p as a key mediator, providing a foundation for further studies on its physiological relevance and regulatory mechanisms.