title: Molecular cloning of rice serotonin N-acetyltransferase, the penultimate gene in plant melatonin biosynthesis.
abstract: 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 cDNAs in Escherichia coli and screened SNAT activity by measuring N-acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N-acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N-acetylserotonin. The values for Km and Vmax were 385 μm 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 mm 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.
authors.affiliation: Department of Biotechnology, Bioenergy Research Center, Chonnam National University, Gwangju, Korea; Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Kiyoon
authors.initials: K
authors.lastname: Kang
authors.forename: Kyungjin
authors.initials: K
authors.lastname: Lee
authors.forename: Sangkyu
authors.initials: S
authors.lastname: Park
authors.forename: Yeong
authors.initials: Y
authors.lastname: Byeon
authors.forename: Kyoungwhan
authors.initials: K
authors.lastname: Back
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D047068:Arylalkylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008550:Melatonin / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013995:Time
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D012701:Serotonin; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin