Browsing by Author "Nourbakhsh, Aida"
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- Characterization of the inositol monophosphatase gene family in ArabidopsisNourbakhsh, Aida; Collakova, Eva; Gillaspy, Glenda E. (Frontiers, 2015-01-09)Synthes is of myo-inositol is crucial in multicellular eukaryote for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca2+ signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase-like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant grow this impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.
- Molecular Characterization of Inositol Monophosphatase Like Enzymes in Arabidopsis thalianaNourbakhsh, Aida (Virginia Tech, 2012-06-11)myo-Inositol synthesis and catabolism are crucial in many multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. myo-inositol monophosphatase (IMP) is a major enzyme required for the synthesis of myo-inositol and breakdown of inositol (1,4,5)-trisphosphate (InsP3), a potent second messenger involved in many biological activities. Arabidopsis contains a single canonical IMP gene, which was previously shown in our lab to encode a bifuntional enzyme with both IMP and L-galactose 1-phosphatase activity. Analysis of metabolite levels in imp mutants showed only slight modifications with less myo-inositol and ascorbate accumulation in these mutants. This result suggests the presence of other functional IMP enzymes in plants. Two other genes in Arabidopsis encode chloroplast proteins, which we have classified as IMP-like (IMPL), because of their greater homology to the prokaryotic IMPs such as the SuhB, and CysQ proteins. Prokaryotic IMP enzymes are known to dephosphorylate D-Inositol 1-P (D-Ins 1-P) and other substrates in vitro, however their in vivo substrates are not characterized. A recent study revealed the ability of IMPL2 to complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, which suggested an important role for IMPL2 in amino acid synthesis. The research presented here focuses on the characterization of IMPL functional roles in plant growth and development. To accomplish this I performed kinetic comparisons of the Arabidopsis recombinant IMPL1 and IMPL2 enzymes with various inositol phosphate substrates and with L-histidinol 1-phosphate, respectively. The data supports that IMPL2 gene encodes an active histidinol 1-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme which has the ability to hydrolyze D-Ins 1-P substrate and may be involved in the recycling of inositol from the second messenger, InsP3. Analysis of metabolite levels in impl2 mutant plants reveals that impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway. Together these data solidify the catalytic role of IMPL2 in histidine synthesis in plants and highlight its importance in plant growth and development.