Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).
The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 μm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event.
Department of Biotechnology, Bioenergy Research Center, Chonnam National University, Gwangju, Korea.
Yeong
Y
Byeon
Department of Biotechnology, Bioenergy Research Center, Chonnam National University, Gwangju, Korea.
Hyoung Yool
HY
Lee
Department of Biotechnology, Bioenergy Research Center, Chonnam National University, Gwangju, Korea.
Kyoungwhan
K
Back
D047068:Arylalkylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008550:Melatonin / Q000096:biosynthesis / Q000235:genetics; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D010940:Plant Proteins; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin
Escherichia coli expression; N-acetylserotonin; SNAT isogene; rice; sheep SNAT; substrate specificity