Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots

:  Rice (Oryza sativa) N‐acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S‐transferase...

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Bibliographic Details
Published in:Journal of pineal research 2013-03, Vol.54 (2), p.139-144
Main Authors: Park, Sangkyu, Byeon, Yeong, Kim, Young-Soon, Back, Kyoungwhan
Format: Article
Language:English
Subjects:
Acetylserotonin O-Methyltransferase - genetics
Acetylserotonin O-Methyltransferase - metabolism
Animals
Chromatography, High Pressure Liquid
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Humans
Kinetics
light effect
melatonin
Melatonin - metabolism
N-acetylserotonin methyltransferase
Oryza - enzymology
Oryza sativa
Plant Shoots - genetics
Plant Shoots - metabolism
Reverse Transcriptase Polymerase Chain Reaction
rice seedlings
Seedlings - enzymology
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Summary::  Rice (Oryza sativa) N‐acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S‐transferase (GST)‐fused recombinant osASMT (GST‐osASMT) and GST‐free osASMT showed specific enzyme activities of 6.6 and 12.6 pmol/min per mg protein, respectively. When evaluated by the Lineweaver‐Burk equation, GST‐free osASMT exhibited a Km of 864 μm. An in vitro enzyme assay of purified osASMT showed melatonin formation to be proportional to the enzyme and substrate concentrations, as well as time. Unlike animal ASMT, high substrate concentrations did not inhibit the activity of osASMT. Finally, melatonin biosynthesis in rice seedlings was affected by light intensity, with etiolated shoots grown in continuous darkness producing more melatonin than shoots grown in continuous light. The level of melatonin in relation to the light intensity closely paralleled the mRNA level of osASMT in the shoots, suggesting that endogenous melatonin is upregulated in darkness, as is the case in animals.
ISSN:0742-3098
1600-079X
DOI:10.1111/j.1600-079X.2012.01019.x