Functional Characterization of GNA1 as a Serotonin N‐Acetyltransferase Reveals a Key Role in the Serotonin to Melatonin Pathway in Saccharomyces cerevisiae

ABSTRACT Melatonin ( N ‐acetyl‐5‐methoxytryptamine) is an evolutionarily conserved molecule with diverse physiological functions across prokaryotes, plants, and animals, including circadian rhythms regulation in animals and developmental modulation in plants. Although the biosynthetic pathways of melatonin have been well elucidated in mammals and plants, the enzymatic mechanisms underlying microbial melatonin synthesis remain largely unexplored. Saccharomyces cerevisiae , a genetically tractable eukaryotic model, provides a valuable system for elucidating fungal melatonin biosynthesis. In this study, we identified the GNA1 gene as a potential serotonin N ‐acetyltransferase candidate in S. cerevisiae through genome‐wide comparative analysis. The GNA1 protein was heterologously expressed in Escherichia coli BL21(DE3), purified, and subjected to detailed enzymatic characterization. In vitro assays revealed that GNA1 exhibits acetyltransferase activity toward both serotonin and 5‐methoxytryptamine (5‐MT), with maximal catalytic efficiency observed at 30°C and pH 8.5. Substrate specificity and kinetic analyses demonstrated a pronounced preference for 5‐MT, supporting a biosynthetic route in which serotonin undergoes O ‐methylation before acetylation by GNA1 to yield melatonin. This study provides the first biochemical evidence linking GNA1 to melatonin biosynthetic pathway in yeast and offers new insights into microbial melatonin biosynthesis, highlighting its potential evolutionary and metabolic significance.