Purification and cloning of a γ-glutamyl transpeptidase from onion ( Allium cepa)

• Published in: Phytochemistry (Oxford) 2005-03, Vol.66 (5), p.515-522
• Main Authors: Shaw, Martin L., Pither-Joyce, Meeghan D., McCallum, John A.
• Format: Article
• Language: English
• Subjects: Alliaceae; Allium cepa; Amino Acid Sequence; amino acid sequences; Biological and medical sciences; chemical structure; Chromatography, Affinity; Cloning; Cloning, Molecular; Consensus Sequence; Enzymes; Enzymology; Fundamental and applied biological sciences. Psychology; gamma-glutamyltransferase; gamma-Glutamyltransferase - genetics; gamma-Glutamyltransferase - isolation & purification; gamma-Glutamyltransferase - metabolism; Glutathione; Humans; Kidney - enzymology; Liliaceae; Metabolism; molecular cloning; Molecular Sequence Data; Molecular Weight; nucleotide sequences; Onion; onions; Onions - enzymology; Onions - genetics; Peptide Fragments - chemistry; Plant physiology and development; plant proteins; Protein Subunits - isolation & purification; Protein Subunits - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; vegetable crops; γ-Glutamyl peptides; γ-Glutamyl transpeptidase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2005.01.017

Onion γ-glutamyl transpeptidase catalyses hydrolysis of γ-glutamyl linkages in γ-glutamyl peptides and transfer of the γ-glutamyl group to amino acids and peptides and has high affinity for glutathione and glutathione conjugates. γ-Glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of γ-glutamyl linkages in γ-glutamyl peptides and transfer of the γ-glutamyl group to amino acids and peptides. Although plant γ-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate γ-glutamyl- p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion γ-glutamyl peptide, γ-glutamyl trans- S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme.