Diversification and evolution of L-myo-inositol 1-phosphate synthase11Dedicated to Dr. Frank Eisenberg, Jr., who introduced A.L.M. to this fascinating enzyme/protein.

AbstractL-myo-Inositol 1-phosphate synthase (MIPS, EC 5.5.1.4), the key enzyme in the inositol and phosphoinositide biosynthetic pathway, is present throughout evolutionarily diverse organisms and is considered an ancient protein/gene. Analysis by multiple sequence alignment, phylogenetic tree generation and comparison of newly determined crystal structures provides new insight into the origin and evolutionary relationships among the various MIPS proteins/genes. The evolution of the MIPS protein/gene among the prokaryotes seems more diverse and complex than amongst the eukaryotes. However, conservation of a ‘core catalytic structure’ among the MIPS proteins implies an essential function of the enzyme in cellular metabolism throughout the biological kingdom

Fructose-1, 6-bisphosphatase in human fetal brain and liver during development

Activity of fructose-1,6-bisphosphatase (EC 3.1.3.11), one of the key gluconeogenic enzymes, was measured in human fetal brain and liver during development. Fructose-1,6-bisphosphatase was distributed throughout the different regions of the brain. In contrast to the partially purified enzyme from the brain, the liver enzyme was dependent on Mg2+ for maximal activity, EDTA, citrate, oleate and linoleate were stimulatory, whereas 5'-AMP inhibited the activity of the liver enzyme

An insight into the molecular basis of salt tolerance of L-myo-inositol 1-P synthase (PcINO1) from porteresia coarctata (Roxb.) tateoka, a halophytic wild rice1

The molecular basis of salt tolerance of L-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1

Modification of brain fructose-1, 6-bisphosphatase activity by chelators: "Induction" of 5'-AMP sensitivity

Brain fructose-1, 6-bisphosphatase (EC 3.1.3.11) from various sources are ordinarily insensitive to 5'-AMP. In addition to stimulation and conferring a "neutral" behaviour, prior treatment with histidine, EDTA or imidazole renders the brain enzyme sensitive to 5'-AMP. The histidine treated enzyme(s) bind to Blue-Sepharose CL-6B column and are specifically eluted by 5'-AMP in contrast to the untreated enzyme(s) which do not bind to the affinity column at all. The histidine effect in inducing 5'-AMP sensitivity was abolished by treatment of the native enzyme by subtilisin or by a number of divalent cations including Zn++

Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: a physiological and proteomic approach

Salinity poses a serious threat to yield performance of cultivated rice in South Asian countries. To understand the mechanism of salt-tolerance of the wild halophytic rice, Porteresia coarctata in contrast to the salt-sensitive domesticated rice Oryza sativa, we have compared P. coarctata with the domesticated O. sativa rice varieties under salinity stress with respect to several physiological parameters and changes in leaf protein expression. P. coarctata showed a better growth performance and biomass under salinity stress. Relative water content was conserved in Porteresia during stress and sodium ion accumulation in leaves was comparatively lesser. Scanning electron microscopy revealed presence of two types of salt hairs on two leaf surfaces, each showing a different behaviour under stress. High salt stress for prolonged period also revealed accumulation of extruded NaCl crystals on leaf surface. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and subsequent quantitative image analysis. Out of more than 700 protein spots reproducibly detected and analyzed, 60% spots showed significant changes under salinity. Many proteins showed steady patterns of up- or downregulation in response to salinity stress. Twenty protein spots were analyzed by MALDI-TOF, leading to identification of 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis and chaperone functions. We hypothesize that some of these proteins confer a physiological advantage on Porteresia under salinity, and suggest a pattern of salt tolerance strategies operative in salt-marsh grasses. In addition, such proteins may turn out to be potential targets for recombinant cloning and introgression in salt-sensitive plants

Further characterization of phosphoinositol kinase isolated from germinating mung bean seeds

Phosphoinositol kinase isolated and purified from germinating mung bean seeds has been further characterized. The rate of phosphorylation varies with different inositol phosphates and this is consistent with the Km and Vmax for each of the substrates. The phosphate transfer from ATP has been found to be mediated by a phosphoprotein intermediate. In a particular step of the reaction the immediate product of the reaction has been found to be most inhibitory, other products being less or non-inhibitory. The inhibition has been found to be competitive in nature. The Kis have been found to range between 0.6 and 1 × 10-4 M. ADP also inhibited non-competitively with respect to IP5. Ki for this has been found to be 2.3 × 10-4 M. The purified enzyme migrated as a single protein band on polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulphate it is dissociated into 3 subunits in the ratio 1 : 1 : 1. The MW of the three subunits are approx. 86 000, 56 000 and 35 000. The MW of the enzyme has been found to be approx. 177 000

The formation of cyclic inositol 1,2-monophosphate, inositol 1-phosphate, and glucose 6-phosphate by brain preparations stimulated with deoxycholate and calcium: a gas chromatographic study

A gas chromatographic method has been developed for the separation and isolation of water-soluble phosphates as trimethylsilyl ethers. With this method cyclic inositol 1,2-monophosphate and inositol 1-phosphate, derived from endogenous phosphatidylinositol, have been shown to increase when a particulate portion of brain homogenate is stimulated with deoxycholate and Ca++, confirming earlier observations of Lapetina and Michell (1). Concomitant with the appearance of inositol phosphates is the stimulated formation of glucose 6-phosphate in the whole homogenate. Although ATP can replace deoxycholate and Ca++ in a dialyzed homogenate, glucose 6-phosphate apparently does not arise by any known metabolic pathway but from another unidentified source

Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase

Chloroplasts from 5 to 7 day old Vigna radiata seedling, grown under alternate light/dark conditions or from green Euglena gracilis Z. cells have been found to harbor L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) activity. In contrast, dark-grown V. radiata seedlings, or streptomycin-bleached Euglena cells exhibit either reduced or no enzyme activity. An apparent enhancement of the chloroplastic inositol synthase by growth in presence of light is observed

Osmolyte regulation in abiotic stress

To withstand osmotic stress induced by salinity, drought or extreme temperatures, all organisms have evolved a machinery to synthesize metabolites, termed "compatible solutes" or "osmo-protectants", which help in raising the osmotic pressure and thereby maintaining both the turgor pressure and the driving gradient for water uptake. In addition, these compounds also help in maintaining the structural integrity of enzymes, membranes and other cellular components during the stress regime. Of special importance among these metabolites is nitrogen containing compounds (e.g., quaternary amino compounds and proline) and hydroxyl compounds (e.g., polyols and oligosaccharides). These compounds are distributed throughout the biological kingdom and are generally products of stress-induced pathway extensions, although normal metabolites such inositols may also act as osmolytes. Chemically, different osmolytes function through a common mechanism of stabilization of proteins under stress or by osmotic adjustments, and these mechanisms seem to be universal among the biological system. Over-expression of genes for the synthesis of different osmolytes in transgenics enables the plants to cope better with the stress due to higher accumulation of the concerned osmolytes. However, in several cases, such as trehalose and inositol, the accumulation is far below the required amount and it is conjectured that these metabolites might function in a manner unrelated to their osmolyte role and are hence more involved in the general growth and development of the plants under abiotic stress conditions