Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT

Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the final two enzymes in the melatonin synthesis pathway in plants. Although their corresponding genes have been cloned, their cellular localization and enzymatic characteristics are unknown. Using confocal microscopy, we showed that SNAT protein is localized in chloroplasts, whereas ASMT is expressed in the cytoplasm. In vitro measurement of ASMT enzyme activity revealed a peak of activity in roots, but SNAT enzyme activity was not detected in any plant tissues. This may be attributed in part to an effect of chlorophyll because SNAT enzyme activity was greatly inhibited by chlorophyll in a dose-dependent manner. Because the SNAT protein of cyanobacteria is thermophilic, we examined the effect of temperature on the activity of the rice SNAT and ASMT enzymes. Purified recombinant rice SNAT and ASMT enzymes had an optimum temperature for activity of 55 degrees C. The K-m and V-max values for SNAT at 55 degrees C were 270m and 3.3nmol/min/mg protein, whereas the K-m and V-max for ASMT were 222m and 9nmol/min/mg protein, respectively. The catalytic efficiency (V-max/K-m) values of SNAT and ASMT were 16-fold and 4054-fold higher at 55 degrees C than at 30 degrees C suggestive of increased melatonin production at high temperature in plants.