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Cloning and functional characterization of a homoglutathione synthetase from pea nodules

AuthorsIturbe-Ormaetxe, Iñaki ; Heras, Begoña; Matamoros Galindo, Manuel Ángel ; Ramos Escribano, Javier ; Morán, José F. ; Becana Ausejo, Manuel
Issue DateMay-2002
PublisherBlackwell Publishing
CitationPhysiologia Plantarum 115 (1): 69-73 (2002)
AbstractThe thiol tripeptide glutathione (GSH; γGlu-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; γGlu-Cys-βAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, 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-1 mg-1 protein with a standard substrate concentration (5 mM β-alanine) and Km values of 1.9 mM for β-alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for β-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.
Description16 Pags.- 2 Figs. The definitive version, with all 3 Figs., is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1399-3054
Publisher version (URL)http://dx.doi.org/10.1034/j.1399-3054.2002.1150107.x
Identifiersdoi: 10.1034/j.1399-3054.2002.1150107.x
issn: 0031-9317
Appears in Collections:(EEAD) Artículos
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