Thesis advisor: Mary F. Roberts / Inositol-containing molecules and inositol phosphatases have diverse roles in cells. One of the inositol phospholipids phosphatases, PTEN (Phosphatase and Tensin Homolog deleted on Chromosome Ten), is a tumor suppressor and antagonizes the PI3K signaling pathway by dephosphorylating PI(3,4,5)P3 at the 3 position of the inositol ring. In testing predictions of a molecular dynamics simulation, a hydrophobic site adjacent to the active site on PTEN was identified and verified by protein kinetic studies. This hydrophobic site plays an important role in substrate and substrate analogue binding with one of residues, Arg47, critical for PTEN phosphatase activity. Mutations of Arg47 reduced enzyme activities toward both the short-chain substrate as monomers and micelles and long-chain phospholipid presented in vesicles. PI(4,5)P2 the product of PI(3,4,5)P3 dephosphorylation, activates PTEN. Studies by others suggested this occurred when the product was bound to the N-terminal region of the protein (not visible in the crystal structure). However, no direct proof of this existed. The effect of PI(4,5)P2 on PTEN enzyme activities in different substrates systems was studied. 31P NMR was used to probe the spatial location and functional role of PI(4,5)P2 binding site. The fixed field 31P NMR and high resolution field cycling 31P NMR results indicated there are discrete sites for both substrate and activator lipids on PTEN, and both of sites are spatially separate from the hydrophobic site. The active site, adjacent hydrophobic site, and N-terminal activator binding site worked synergistically to regulate PTEN interacting with the membrane. Thermophilic and hyperthermophilic archaea and bacteria thrive at high temperatures. They often accumulate small organic molecules, called compatible solutes or osmolytes, to protect proteins from thermal denaturation. The thermoprotection mechanism of compatible solutes was explored using inositol monophosphatase (IMPase) from Archaeoglobus fulgidus as the model protein. The protective effect of unusual compatible solutes, di-inositol-1,1'-phosphate (DIP) and diglycerol phosphate (DGP), as well as common compatible solutes glutamate and other anions, on the IMPase thermostability was studied. Specific binding sites of glutamate ions on the IMPase were identified by crystallography and field cycling NMR. However, mutations at these discrete binding sites did not eliminate the thermoprotection, but reduced the thermal stability (Tm) of the protein. Our results indicate the specific binding of osmolytes to the protein exists, but they do not account for the thermoprotection. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Crystallization studies in presence of organic osmolytes were conducted to better understand the specific mechanism of compatible solute binding to the inositol monophosphatase of Archaeoglobus fulgidus. The synthesis of a-diglycerol phosphate, one of the natural osmolytes of A. fulgidus, was also completed for kinetic testing of its I-1-Pase thermoprotective properties and for crystallization trials. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Inositol is important for normal growth and development in plants. The regulation of the inositol biosynthetic enzymes, <I>myo</I>-inositol phosphate synthase (MIPS) and <I>myo</I>-inositol monophosphatase (IMP) was investigated. The specific aims of this research were (1) to develop a tool to study MIPS protein accumulation in a model plant system, <I>Arabidopsis thaliana (At)</I> and potentially other plant species and (2) to determine the spatial expression patterns of <I>Lycopersicon esculentum</I> IMP-2 (<I>Le</I>IMP-2) at the cellular level. <I>Myo-inositol phosphate synthase (mips)</I> genes have been identified in plants, animals, fungi and bacteria. Alignment of the predicted amino acid sequences of <I>At</I>MIPS-1, -2 and <I>Glycine max</I> MIPS (<I>Gm</I>MIPS) indicated that <I>At</I>MIPS-1 and <I>Gm</I>MIPS are 87% identical, and <I>At</I>MIPS-2 and <I>Gm</I>MIPS are 89% identical. Based on these data, a <I>Gmmips</I> cDNA was fused at the N-terminus to a 6X histidine tag (5' GAC GAC GAC GAC GAC GAC 3'), cloned into an overexpression vector and overexpressed in <I>E. coli</I>. The fusion protein, HISMIPS, was extracted using denaturing conditions and purified using Ni²⁺ affinity chromatography. Anti-<I>Gm</I>MIPS antiserum from rabbit detected the recombinant HISMIPS protein (76 kD), and <I>Gm</I>MIPS (64 kD). Affinity purification by subtractive chromatography yielded anti-<I>Gm</I>MIPS antibody that detected <At</I>MIPS (66 kD) and a protein (34 kD) of unknown function. <I>At</I>MIPS accumulated to high levels in unopened flowers, opened flowers, and immature siliques (6 mm in length or less), but was not detectable in bolts, cauline or rosette leaves. The tomato <I>inositol monophosphatase (Leimp)</I> genes are a developmentally regulated multigene family. From analysis of sequences, <I>Leimp</I>-2 is intron-less and has the putative start site of translation located at +108 bp downstream from the putative start site of transcription. Investigation of the 5' UTR revealed the 3' end of a partial open reading frame (338 bp) highly homologous to the gene for calmodulin. Three light responsive elements and a cold responsive element were also identified in the 5' UTR. Transgenic <I>Leimp</I>-2::<I>uid</I>A plants were produced using the existing construct of the <I>Leimp</I>-2 promoter fused to the <I>uid</I>A gene (J. Keddie, University of California at Berkeley). Seedlings were preserved and sectioned. Using histological techniques, the analysis of the <I>Leimp</I>-2 promoter::<I>uid</I>A transgenic seedlings revealed that the <I>Leimp</I>-2 promoter causes expression at the base of the shoot apex and within leaflets of the first set of fully expanded leaves. Further, <I>Leimp</I>-2 promoter expression was localized to epidermal and cortex cells on the abaxial side of the 1st and 2nd fully expanded compound leaves. These studies of MIPS and IMP expression lay a foundation for a better understanding of the regulation of inositol biosynthesis in Arabidopsis, tomato, and other plant species. / Master of Science

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. / Ph. D.