Crystal Structure of Inositol 1-Phosphate Synthase from Mycobacterium tuberculosis, a Key Enzyme in Phosphatidylinositol Synthesis

Norman, Richard A.; McAlister, M.; Murray‐Rust, Judith; Movahedzadeh, Farahnaz; Stoker, Neil G.; McDonald, Neil Q.

doi:10.1016/s0969-2126(02)00718-9

Cited by 59 publications

(86 citation statements)

References 33 publications

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“…Until now, over 60 genes homologous to INO1 have been cloned and characterized from a wide variety of prokaryotic, archaeal, and eukaryotic sources (9 -12), and the conservation of a probable "core catalytic structure" among all has been proposed (12). The crystal structures of MIPS(s) from Saccharomyces and Mycobacterium have been worked out, providing evidence for the structural insight for the proposed reaction mechanism (13)(14)(15)(16)(17).…”

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confidence: 99%

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Majee

Maitra

Dastidar

et al. 2004

Journal of Biological Chemistry

168

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L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174 -210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200 -300 mM NaCl with retention of ϳ40 -80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.Inositols are six-carbon cyclohexane hexitols found ubiquitously in the biological kingdom, and its metabolism plays a vital role in growth regulation, membrane biogenesis, osmotolerance, and in many other processes. As phosphorylated derivatives, its role as a phosphorus store and as a "second messenger" in signal transduction pathways has long been recognized. myo-Inositol, physiologically the most favored stereoisomer among the eight possible geometric isomers of inositol, also enters into an array of biochemical reactions having diverse functions in cellular metabolism both as free and conjugated and phosphorylated or methylated forms (1-3).The primary enzyme for the synthesis of L-myo-inositol 1-phosphate from glucose 6-phosphate is L-myo-inositol-1-phosphate synthase (EC 5.5.1.4; referred to as MIPS), 1 which synthesizes L-myo-inositol 1-phosphate through an internal oxidoreduction reaction involving NAD ϩ . Free inositol is generated by dephosphorylation of the MIPS product by a specific Mg 2ϩ -dependent inositol-1-phosphate phosphatase (EC 3.1.3.25). This mechanism is followed by all myo-inositolproducing organisms throughout the phylogenetic lines, and MIPS has been identified as an evolutionarily conserved protein (4). The structural gene coding for cytosolic MIPS, termed INO1, was first identified in yeast, Saccharomyces cerevisiae (5, 6) and cloned by Klig and Henry (7)....

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confidence: 99%

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Majee

Maitra

Dastidar

et al. 2004

Journal of Biological Chemistry

168

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“…Figure 1 shows that the four highly conserved functional sequences are present in the Pv_BAT93 MIPS sequence. These four domains are involved in MIPS protein binding and are essential for MIPS functions, such as cofactor NAD + binding and reaction catalysis (Majumder et al 1997;Norman et al 2002). Figure 1 also predicted a conserved transmembrane motif (CEDSLLAAPIILDLVLLAELSTR), located about 68 amino acids from the C-terminus in both P. vulgaris and other species.…”

Section: Characterization Of the Deduced Pv_bat93 Mips Proteinmentioning

confidence: 99%

Comparative Expression and Cellular Localization of Myo-inositol Phosphate Synthase (MIPS) in the Wild Type and in an EMS Mutant During Common Bean (Phaseolus vulgaris L.) Seed Development

et al. 2011

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In plants, the myo-inositol-1-phosphate synthase (EC 5.5.1.4; MIPS) is an evolutionarily conserved enzyme protein and catalyzes the synthesis of myo-inositol, which is a central molecule required for cell metabolism and plant growth. A full-length cDNA encoding common bean (Phaseolus vulgaris L.) MIPS (designated Pv_BAT93 MIPS) was isolated from seed of P. vulgaris (cv, BAT93) by rapid amplification of cDNA ends. The cDNA of Pv_BAT93 MIPS comprised 1,873 bp, which encodes 510 amino acids. The Pv_BAT93 MIPS gene was highly homologous with those of other plant species, such as P. vulgaris (cv, Taylor's Horticultural), Arabidopsis thaliana, Medicago sativa and Glycine max. DNA blot analysis indicated that at least three copies of MIPS are present in the common bean genome. RT-PCR analysis indicated that the Pv_BAT93 MIPS transcripts existed in developing seeds and other common bean tissues, including leaves, flowers, and cotyledons but showed lower levels expression in roots and stems. Real-time quantitative PCR showed that Pv_BAT93 MIPS gene is differentially expressed during common bean seed development. In situ hybridization of developing seeds in the wild type and in the ethyl methanesulfonate mutant showed that expression of Pv_BAT93 MIPS was identified in the embryo tissues, from the globular to late cotyledon stages, confirming a possible involvement of Pv_BAT93 MIPS in common bean seed development.

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“…The archaeal mIPS did not have divalent cations present in the structure, although in two of the four subunits there was one metal ion whose properties were consistent with K ϩ that interacted with NAD ϩ . A metal ion at this position corresponds to the Zn 2ϩ tentatively identified in structures of M. tuberculosis mIPS (15) and more recently in the yeast IPS (16). However, electrostatic field calculations carried out for the A. fulgidus mIPS suggested that a second metal ion-binding site might be located close to the site occupied by K ϩ (13).…”

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confidence: 90%

“…NAD ϩ binding to vacant sites decreases the intrinsic fluorescence intensity but not the wavelength of maximum emission of the archaeal mIPS protein. This change in fluorescence intensity can be used to quantify NAD ϩ binding to the proteins by measuring the intrinsic fluorescence intensity at 334 nm compared with that for the protein where only buffer was added over an NAD ϩ concentration range of [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] M. There are two tryptophans near the active site of A. fulgidus mIPS (13) that could have altered characteristics upon NAD ϩ binding. From the change in fluorescence intensity as a function of added NAD ϩ , we used the concentration for 50% of the maximum change to define an apparent K D for NAD ϩ binding (see Fig.…”

Section: Figmentioning

confidence: 99%

“…Despite significant differences in protein length and sequence, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae (14) and bacterial mIPS from Mycobacterium tuberculosis (15). The archaeal mIPS did not have divalent cations present in the structure, although in two of the four subunits there was one metal ion whose properties were consistent with K ϩ that interacted with NAD ϩ .…”

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confidence: 97%

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Probing the Mechanism of the Archaeoglobus fulgidus Inositol-1-phosphate Synthase

Neelon

Wang

Stec

et al. 2005

Journal of Biological Chemistry

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myo-Inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate. In the sulfate-reducing archaeon Archaeoglobus fulgidus it is a metal-dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-inositol-1,1 -phosphate. Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS. Six charged residues in the active site (Asp 225 , Lys 274 , Lys 278 , Lys 306 , Asp 332 , and Lys 367 ) and two noncharged residues (Asn 255 and Leu 257 ) have been changed to alanine. The charged residues are located at the active site and were proposed to play binding and/or direct catalytic roles, whereas noncharged residues are likely to be involved in proper binding of the substrate. Kinetic studies showed that only N255A retains any measurable activity, whereas two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD ؉ to NADH. The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31 P line width of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD ؉ (detected via changes in the protein intrinsic fluorescence). Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.myo-Inositol-1-phosphate synthase (mIPS) 1 catalyzes the conversion of D-glucose-6-phosphate to L-myo-inositol-1-phosphate, the first step in de novo biosynthesis of myo-inositol. Compounds containing inositol are critical components of signal transduction pathways (1), cell walls in some pathogenic bacteria (2), and various stress responses (3, 4). In some hyperthermophilic archaea and bacteria, inositol is used for the synthesis of unique solutes accumulated to balance external osmotic pressure and protect intracellular macromolecules from heat and/or salt shock (for reviews of osmolytes in hyperthermophiles see Ref. 5). The solute di-myo-inositol-1,1Ј-phosphate (DIP) has been detected in Archaeoglobus fulgidus (6), Methanococcus igneus (7), Pyrococcus sp. (8, 9), and several bacteria of the Thermotogales family when the cells are grown at temperatures above 80°C (10). The DIP synthesized by M. igneus is chiral and composed of L-I-1-P units that are synthesized by mIPS (11). Because the archaeal mIPS enzymes commit significant cell resources to DIP synthesis, the production of DIP must be tightly regulated.The mIPS gene has been identified in the A. fulgidus genome, and the gene product was heterologously expressed in Escherichia coli (12). The recombinant A. fulgidus mIPS is an extremely heat-stable tetramer of 44-kDa subunits (much smaller than the eukaryotic homolog that is a tetramer of 60-kDa subunits). The catalytic activity of the enzyme was also shown to be absolutely dependent on divalent metal ions (Zn 2ϩ , Mn 2ϩ , or Mg 2ϩ ...

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Crystal Structure of Inositol 1-Phosphate Synthase from Mycobacterium tuberculosis, a Key Enzyme in Phosphatidylinositol Synthesis

Cited by 59 publications

References 33 publications

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Comparative Expression and Cellular Localization of Myo-inositol Phosphate Synthase (MIPS) in the Wild Type and in an EMS Mutant During Common Bean (Phaseolus vulgaris L.) Seed Development

Probing the Mechanism of the Archaeoglobus fulgidus Inositol-1-phosphate Synthase

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