Biochem. J. (2005) 391
(567–574) (Printed in Great Britain)
Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays)
Mark SKIPSEY*1, Benjamin G. DAVIS† and Robert EDWARDS*
*Crop Protection Group, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, U.K., and †Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
Unlike animals which accumulate glutathione (g-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or g-glutamyl-L-cysteinyl-b-alanine)], wheat (hydroxymethylglutathione or g-glutamyl-L-cysteinyl-serine) and maize (g-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into g-Glu-Cys. The soya bean enzyme primarily incorporated b-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and a-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor.
Key words: glutathione synthetase, Glycine max (soya bean), homoglutathione synthetase, hydroxymethylglutathione, Triticum aestivum (wheat), Zea mays (maize).
Abbreviations used: AABA, BABA and GABA, a-, b- and g-aminobutyric acid respectively; ACV synthetase, L-d-(a-aminoadipoyl)-L-cysteinyl-D-valine synthetase; BAIBA, b-aminoisobutyric acid; g-EC, g-glutamyl-L-cysteine; g-ECE, g-glutamyl-L-cysteine-glutamic acid; g-ECS, g-glutamyl-L-cysteine synthetase; ESI, electrospray ionization; GS, glutathione synthetase; hGS, homoglutathione synthetase; hGSH, homoglutathione; hmGSH, hydroxymethylglutathione; TOF, time-of-flight.
1To whom correspondence should be addressed (email mark.skipsey@durham.ac.uk).
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AJ272035 (GmGS), AJ579380 (TaGS1), AJ579381 (TaGS2), AJ579382 (TaGS3) and AJ579383 (ZmGS).
Received 3 May 2005/29 June 2005; accepted 11 July 2005
Published as BJ Immediate Publication 11 July 2005, doi:10.1042/BJ20050718
The Biochemical Society, London ©2005
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