SerotoninN-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. TheEscherichia coliorthologue of archaeal SNAT fromThermoplasma volcaniumwas identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be anN-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, withKmandVmaxvalues of 531 μM and 528 pmol/min/mg protein toward serotonin and 201 μM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to therimImutantE. colistrain that showed no growth defect, theEcRimIoverexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. TheEcRimIoverexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect ofEcRimIoverexpression was also observed under cadmium stress. The higher growth rate associated withEcRimIexpression was attributed to increased proteinN-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.