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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS APPROACHES FOR IMPROVING NUTRITIONAL QUALITY OF FOOD CROP SPECIES Title: Tomato y-glutamilhydrolases: expression, characterization, and evidence for heterodimer formation

Authors
item Akhtar, Tariq - UNIV. OF FLORIDA
item Naponelli, Valeria - UNIV. OF FLORIDA
item Gregory, Jesse - UNIV. OF FLORIDA
item Giovannoni, James
item Hanson, Andrew - UNIV. OF FLORIDA

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 3, 2008
Publication Date: December 29, 2008
Citation: Akhtar, T., Naponelli, V., Gregory, J., Giovannoni, J.J., Hanson, A. 2008. Tomato y-glutamilhydrolases: expression, characterization, and evidence for heterodimer formation. Plant Physiology. 148:775-784.

Interpretive Summary: Tetrahydrofolate (THF) and its one-carbon (C1) substituted forms (collectively termed folates) are cofactors in one-carbon transfer reactions that form a number of amino acids, purines, thymidylate,pantothenate, and formyl-methionyl tRNA in nearly all organisms. Folates are necessary nutrients in the human diet and deficiencies can cause major health probles including neural tube defects of newborns. Folates generally have a short, y-linked polyglutamyl tail that affects their biological activity. Folate-dependent enzymes typically prefer polyglutamates whereas folate transporters prefer non-glutamylated forms. In addition, folates are less prone to oxidative breakdown when protein-bound than when free. Polyglutamylation thus tends to enhance cofactor efficacy, to favor folate retention in cells or subcellular compartments, and to protect folates from breakdown. Folates typically have y-linked polyglutamyl tails that make them better enzyme substrates, and worse transport substrates, than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme y glutamyl hydrolase (GGH). We have clones and characterized expression of a family of GGH genes expressed in tomato fruit. Tomato and many fruits lose folate as they mature and thus we are studying genes involved in folate metabolism to better understand how fruit may be bred or engineered to enhance their capacity as sources of dietary folates.

Technical Abstract: It is known that GGH is active as a dimmer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in-vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When co-expressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, while LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed ~95% reduction in folate polyglutamates but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously, and to have catalytic characteristics midway between those of the corresponding homodimers.

Last Modified: 11/23/2014
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