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Molecular cloning of rice serotonin N ‐acetyltransferase, the penultimate gene in plant melatonin biosynthesis
Author(s) -
Kang Kiyoon,
Lee Kyungjin,
Park Sangkyu,
Byeon Yeong,
Back Kyoungwhan
Publication year - 2013
Publication title -
journal of pineal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.881
H-Index - 131
eISSN - 1600-079X
pISSN - 0742-3098
DOI - 10.1111/jpi.12011
Subject(s) - melatonin , enzyme , biology , acetyltransferase , biochemistry , biosynthesis , serotonin , escherichia coli , microbiology and biotechnology , gene , acetylation , receptor , endocrinology
Because of the absence of an arylalkylamine N ‐acetyltransferase ( AANAT ) homolog in the plant genome, the proposal was made that a GCN5 ‐related N ‐acetyltransferase superfamily gene ( GNAT ) could be substituted for AANAT . To clone rice serotonin N ‐acetyltransferase ( SNAT ), we expressed 31 rice GNAT cDNA s in E scherichia coli and screened SNAT activity by measuring N ‐acetyltryptamine after application with 1 m m tryptamine. GNAT 5 was shown to produce high levels of N ‐acetyltryptamine in E . coli , suggesting a possible rice SNAT . To confirm SNAT activity, the GNAT 5 protein was purified through affinity purification from E . coli culture. The purified recombinant GNAT 5 showed high SNAT enzyme activity catalyzing serotonin into N ‐acetylserotonin. The values for K m and V max were 385 μ m and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N ‐acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 m m serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N ‐acetylserotonin methyltransferase ( ASMT ), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate‐limiting enzyme of melatonin biosynthesis in plants.