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Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT
Author(s) -
Byeon Yeong,
Lee Hyoung Yool,
Lee Kyungjin,
Park Sangkyu,
Back Kyoungwhan
Publication year - 2014
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.12103
Subject(s) - enzyme , biochemistry , chemistry , enzyme assay , microbiology and biotechnology , biology
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°C. The K m and V max values for SNAT at 55°C were 270 μ m and 3.3 nmol/min/mg protein, whereas the K m and V max for ASMT were 222 μ m and 9 nmol/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°C than at 30°C suggestive of increased melatonin production at high temperature in plants.