Journal
JOURNAL OF PINEAL RESEARCH
Volume 61, Issue 2, Pages 198-207Publisher
WILEY
DOI: 10.1111/jpi.12339
Keywords
Escherichia coli expression; N-acetylserotonin; rice; sheep SNAT; SNAT isogene; substrate specificity
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Funding
- National Research Foundation of Korea [2014R1A2A1A11050083]
- Basic Research Program [2010-0020141]
- National Research Foundation of Korea [2014R1A2A1A11050083] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 SNATRNAi 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 SNATRNAi 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 K-m and V-max values of the purified recombinant OsSNAT2 were 371m and 4700pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45 degrees C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The invitro 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.
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