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Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay

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Abstract

Genes encoding phytochelatin (PC) synthase have been found in higher plants, fission yeast and worm. Recently, kinetic and mutagenic analyses of recombinant PC synthase have been revealing the molecular mechanisms underlying PC synthesis, however, a conclusive model has not been established. To clarify the mechanism of PC synthase found in eukaryotes, we have compared the two-step reactions catalyzed by the prokaryotic Nostoc PC synthase (NsPCS) and the eukaryotic Arabidopsis PC synthase (AtPCS1). Comparative analysis shows that in the first step of PC synthesis corresponding to the cleavage of γ-glutamylcysteine (γ-EC) from glutathione (GSH), free GSH or PCs acts as a donor molecule to supply a γ-EC unit for elongation of the PC chain, and heavy metal ions are required to carry out the cleavage. Furthermore, functional analyses of various mutants of NsPCS and AtPCS1, selected by comparing the sequences of NsPCS and AtPCS1, indicate that the N-terminal region (residues 1–221) in AtPCS1 is the catalytic domain, and in this region, the Cys56 residue is associated with the PC synthesis reaction. These results enable us to propose an advanced model of PC synthesis, describing substrate specificity, heavy metal requirement, and the active site in the enzyme.

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Abbreviations

PC :

Phytochelatin

AtPCS1 :

Arabidopsis PC synthase

NsPCS :

Nostoc PC synthase

GS H :

Glutathione

γ EC :

γ-Glutamylcystaine

β ME :

β-Mercaptoethanol

IPTG :

Isopropyl-ß-D-thiogalactopyranoside

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Acknowledgements

We would like to thank Professor Meihart H. Zenk (Biozentrum Universität Halle, Germany) for his precious comments and PCs from S. cucubalus, Dr. Matjaz Oven (Leibniz-Institut für Pflanzenbiochemie, Germany) for helpful suggestions, the Arabidopsis Biological Resource Center (Columbus, OH) for libraries and E. coli genetics stock center (Yale University) for E. coli strain ΔzntA RW3110. This work was supported by the Grants-in-Aid for Scientific Research (No. 15310054 and No. 14406031) from the Ministry of Education, Culture, Sports, Science and Technology.

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Authors and Affiliations

  1. Environmental Biotechnology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 1-6 Yamadaoka, Osaka, 565-0871, Japan

    Naoki Tsuji, Osamu Iwabe, Kazuhisa Miyamoto & Kazumasa Hirata

  2. School of Materials Science, Japan Advanced Instituted of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, 923-1292, Japan

    Shingo Nishikori, Sachiko Matsumoto, Kentaro Shiraki & Masahiro Takagi

  3. The Kansai Electric Power Co, Technical Research Center, Amagasaki, Hyogo, 671-0974, Japan

    Hitoshi Miyasaka

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  1. Naoki Tsuji
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  2. Shingo Nishikori
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  3. Osamu Iwabe
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  4. Sachiko Matsumoto
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  5. Kentaro Shiraki
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  8. Kazuhisa Miyamoto
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  9. Kazumasa Hirata
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Correspondence to Kazumasa Hirata.

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Tsuji, N., Nishikori, S., Iwabe, O. et al. Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay. Planta 222, 181–191 (2005). https://doi.org/10.1007/s00425-005-1513-9

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