Biosynthesis of Phytic Acid

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

Although the presence of phytic acid in plant cells has been known for over a century, attempts to determine its biosynthesis in whole plants, plant organs, subcellular organelles, and cell cultures [14,77,78,83], have only recently seen signs of pro- gress. Evidence suggests that phytic acid biosyn- thesis occurs in cisternal endoplasmic reticulum and the product is subsequently deposited in phytin granules [84]. Investigations in duckweed, Spirodela polyrhiza [16] and the slime mold, Dic- Fig. 9. Conformational structures of phytic acid: 1 the 1ax5eq form pH 1 – 9. 2 The 5ax1eq form \ pH 9.5. tyostelium [85,86], provided strong evidence for sequential phosphorylation of Ins3P 1 . Spirodela polyrrhiza: Ins3P 1 “ Ins3,4P 2 “ Ins3,4,6P 3 “ Ins3,4,5,6P 4 “ Ins1,3,4,5,6P 5 “ InsP 6 Dictyostelium: Ins3P 1 “ Ins3,6P 2 “ Ins3,4,6P 3 “ Ins1,3,4,6P 4 “ Ins1,3,4,5,6P 5 “ InsP 6 Both pathways begin with Ins3P 1 which makes biosynthetic sense in that Ins3P 1 is product of both Ins3P 1 synthase and MI kinase. The close relationship between Ins3P 1 formation and phytic acid biosynthesis in developing seeds is revealed in the elegant work of Yoshida et al. [35]. As discussed earlier in regard to Ins3P 1 biosyn- thesis, in situ hybridization of developing rice grains seeds showed that the transcript for Ins3P 1 synthase RINO1 first appeared in the upper half of the embryo two days after anthesis, increased for the next five days and then gradually decreased. Phytin-containing globoids first ap- peared in the scutellum and the aleurone layer four days after anthesis and increased gradually in both tissues. The appearance of globoids coincided with the localization of RINO1 transcripts thereby suggesting that enhanced Ins3P 1 formation drives phytic acid biosynthesis. It is interesting that phosphorylation at the 1 position of InsP n occurs quite late in both biosynthetic pathways suggesting that the second messenger Ins1,4,5P 3 pathway and the phytic acid biosynthetic pathway do not intersect in these tissues. In contrast, phos- pholipase C-triggered production of Ins1,4,5P 3 and subsequent conversion to phytic acid, which is involved in mRNA transport, have been shown in yeast [87]. In all pathways proposed, the 2 position of InsP n is the last position to be phosphorylated.

9. Hydrolysis of phytic acid: