Structure and conformation of phytic acid

Fig. 8. Biochemical conversion of myo-inositol to pinitol. Bracket indicates theoretical intermediate. cal to Ins3P 1 synthase from other plants. Under salinity stress, the Inps 1 RNA up-regulated five-fold or more and free MI accumulated ten-fold [23]. The second step, an O-methyl transferase, methy- lates C4 of MI. Transgenic tobacco plants bearing IMT1, the O-methyl transferase from ice plant, were found to accumulate ononitol and provide better protection under drought and salt-stress conditions than wild-type plants [68]. The enzyme catalyzing step three, epimerization of C1 of onon- itol, has yet to be examined but it is assumed that this step is not rate-limiting. Immunocytology and solute measurements [69] led to discovery of in- creased phloem transport of MI accompanied by increased transport of Na + and inositol to leaves of ice plant under stress. It was found that seedlings of ice plants, which are not salt-tolerant, developed patterns of gene expression and polyol accumulation observed in mature salt-tolerant plants and that MI-enhanced Na + uptake and transport increased [38]. From these data it is proposed that a Na + MI symport might exist that promotes Na + uptake in the ice plant. This is a novel idea which, as the authors point out, may be a general mechanism of controlling Na + uptake in glycophytes. It also offers new clues regarding the osmoregulatory role of O-methyl inositols. It is safe to assume that research on inositol- linked, stress-related processes in plants is still in its pioneering stages. There is an interesting obser- vation that sap collected during the dormant pe- riod winter and early spring from sugar maple Acer saccharum is rich in quebrachitol 1L -2-O- methyl-chiro-inositol, ranging from 4 – 6 of total solids. Yet when trees break dormancy virtually no quebrachitol remains in the sap [70]. Current studies on the role of cyclitols as stable organic osmolytes in trees may be one explanation [65].

7. Structure and conformation of phytic acid

In 1872, Pfeffer showed that subcellular parti- cles in wheat endosperm contained a calciummag- nesium salt of organic phosphate. Two structures were proposed for phytic acid and it took over 50 years to resolve the structural issue. In 1908, Neu- berg proposed a structure that contained three cyclic pyrophosphate moieties and in 1914, Ander- son proposed a structure in which the six hydroxyl groups on MI are esterified with orthophosphate moieties [15,57,71 – 73]. NMR spectroscopy finally resolved the choice in favor of the latter in 1969 [74]. In foods and feed, phytic acid reduces the bioavailibility of inositol, phosphorus, and essen- tial minerals by forming non-assimilable salt com- plexes which lead to detrimental nutritional effects on human and animals [75,76]. Moreover, disposi- tion of this unabsorbed phytate creates environ- mental phosphorus contamination, an agricultural situation that has prompted governmental legisla- tion in Europe and the USA [76]. To meet this challenge, agricultural interests have actively en- couraged development of plant cultivars with re- duced phytic acid as well as related methods of modifying foods and feeds to render their phytate content more nutritional [77]. Cellular roles proposed for phytic acid including inhibition of protein phosphatases and subsequent modulation of calcium channel activity, attenua- tion of endocytosis, and inhibition of clathrin assembly [78,79]. Here, further work is needed to fully confirm these functions. Although the orientation of the phosphate groups in phytic acid was established, there was much debate about the conformation adopted [71,80]. 1ax5eq and 5ax1eq Fig. 9 differ signifi- cantly in overall molecular shape and the orienta- tion of polar groups. Consequently, the two conformations exhibit significant differences in chelating ability, including interaction with proteins. Therefore, the conformational preference of phytic acid in different environments is critical to understanding the chemistry and biochemistry of phytic acid. Assignment of conformations for phytic acid [71] has only recently been fully re- solved. 1 H-NMR spectroscopic methods [80] en- abled Barrientos and Murthy [81] to show that MI adopts the sterically favorable 1ax5eq conforma- tion one phosphate axial and five equatorial in pH range 1.0 – 9.0 and the sterically hindered 5ax 1eq conformation above pH 9.5 Fig. 9. Between pH 9.0 – 9.5 the pKa region of the three least acidic protons both conformations are in dynamic equilibrium. Dynamic NMR indicates that the activation energy for the conformational inversion process is 54.8 9 0.8 kJmole compared to that for ring inversion of cyclohexane, about 45 kJ mole [82]. Inversion to the 5ax1eq form is facili- tated by complexation with metal ions which reduces electrostatic repulsion and thereby stabi- lizes the sterically hindered, dodeca-anionic form. Stabilization of the 5ax1eq form is influenced by the size of the counter ion. Alkali metals Na + , K + , Rb + and Cs + with hydrated radii less than or equal to 2.76 A , stabilize the 5ax1eq form whereas Li + ion hydrated radii 3.4 A , does not stabilize that form. In the presence of larger coun- ter ions such as tetramethylammonium and tetra- butylammonium ions, the presence of the sterically hindered 5ax1eq form is not observed, thus indi- cating that complexation with counter ions is es- sential for ring inversion of phytic acid [82]. NMR spectroscopy also indicated that the con- formational preferences of individual isomers at different pH’s are dictated by structural features unique to the isomer such as the number of phos- phate moieties and the regiochemical and stereo- chemical arrangement of the phosphates [81]. Ins1,2,3,4,6P 5 adopts the 1ax5eq form in the pH range 1.0 – 9.0 and above 9.5 both 1ax5eq and 5ax1eq forms exist in dynamic equilibrium; the exclusive presence of the 5ax1eq form is not observed. Inositol phosphates containing less than 5 phosphates showed no proclivity to undergo ring inversion to the sterically hindered form [81]. Molecular modeling studies were carried out using ab initio, semi-empirical and force field methods MacroModel V6.0 and Gaussian 94, for both the 1ax5eq and 5ax1eq conformations of phytic acid in the fully protonated and dodeca-an- ionic state [82]. Molecular modeling calculations were consistent with NMR results in aqueous so- lution. Interestingly, calculations predicted that the relative stability of the two conformations is the same in vacuum and aqueous solution, namely, in the fully protonated state the sterically favourable 1ax5eq form of phytic acid is more stable than the 5ax1eq form and that in the dodeca-anionic state the sterically hindered 5ax 1eq form is more stable than the 1ax5eq form [82].

8. Biosynthesis of Phytic Acid