Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production inSaccharomyces cerevisiae,PAA1, a polyamine acetyltransferase, a homolog of the vertebrate’s aralkylamineN-acetyltransferase (AANAT). In this study, we assessed the in vivo function ofPAA1by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for newN-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT inS. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression inE. colibecause, curiously, this system evidenced higher differences than the overexpression in their own hostS. cerevisiae.Our results confirm thatPAA1possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detectedHPA2as a new arylalkylamineN-acetyltransferase inS. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.