Cloning and functional characterization of a homoglutathione synthetase from pea nodules
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Abstract
<jats:p>The thiol tripeptide glutathione (GSH; <jats:italic>γ</jats:italic>Glu‐Cys‐Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; <jats:italic>γ</jats:italic>Glu‐Cys‐<jats:italic>β</jats:italic>Ala), in addition to or instead of GSH. We have isolated from a pea (<jats:italic>Pisum sativum</jats:italic> L.) nodule library a cDNA, <jats:italic>GSHS2</jats:italic>, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min<jats:sup>−1</jats:sup> mg<jats:sup>−1</jats:sup> protein with a standard substrate concentration (5 m<jats:italic>M β</jats:italic>‐alanine) and K<jats:sub>m</jats:sub> values of 1.9 m<jats:italic>M</jats:italic> for <jats:italic>β</jats:italic>‐alanine and 104 m<jats:italic>M</jats:italic> for glycine. The specificity constant (V<jats:sub>max</jats:sub>/K<jats:sub>m</jats:sub>) shows that the pure enzyme is 57.3‐fold more specific for <jats:italic>β</jats:italic>‐alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.</jats:p>
Journal
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- Physiologia Plantarum
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Physiologia Plantarum 115 (1), 69-73, 2002-05
Wiley
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Details 詳細情報について
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- CRID
- 1360292621399905408
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- NII Article ID
- 30013422791
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- NII Book ID
- AA00774354
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- ISSN
- 13993054
- 00319317
- http://id.crossref.org/issn/00319317
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- Data Source
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- Crossref
- CiNii Articles