title: Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles.
abstract: gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.
authors.affiliation: Horticultural Sciences and Food Science Department, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Orsomando
authors.forename: Rocío Díaz
authors.initials: RD
authors.lastname: de la Garza
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Green
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Peng
authors.forename: Philip A
authors.initials: PA
authors.lastname: Rea
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Ryan
authors.forename: Jesse F
authors.initials: JF
authors.lastname: Gregory
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D027461:Beta vulgaris / Q000378:metabolism; D032461:Chromosomes, Plant; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020224:Expressed Sequence Tags; D005492:Folic Acid / Q000737:chemistry*; D017343:Genes, Plant; D005971:Glutamates / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D018532:Pisum sativum / Q000378:metabolism; D011099:Polyglutamic Acid / Q000737:chemistry*; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D013763:Tetrahydrofolates / Q000494:pharmacology; D013997:Time Factors; D014461:Ultracentrifugation; D014617:Vacuoles / Q000378:metabolism*; D011623:gamma-Glutamyl Hydrolase / Q000737:chemistry / Q000502:physiology*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005971:Glutamates; D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; D005492:Folic Acid; D010450:Endopeptidases; D011623:gamma-Glutamyl Hydrolase; C005984:5-methyltetrahydrofolate