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:
The enzymes responsible for phytic acid hydrol- ysis are phytases, a special class of phosphatases
that catalyze the sequential hydrolysis of phytic acid [14,15]. The sequence of hydrolysis by acid
phytases was first established by Tomlinson and Ballou [88,89]. Their methods for determining the
structures of intermediate inositol phosphates are still widely used. Based on the position of the first
phosphate hydrolyzed, two classes of acid phytases are recognized, the 6-phytase EC 3.1.3.26 and
the 3-phytase EC 3.1.3.8 [15]. Both hydrolyze phytic acid to Ins2P
1
. The X-ray crystal structure of a 3-phytase from Aspergillus niger has been
determined [90]. The structure consists of a large ab-domain, which shows structural similarity to a
high molecular weight acid phosphatase in rats, and a smaller a-domain. The active site contains
the amino acid sequence, RHGXRXP, common in many acid phosphatases, and a cluster of basic
amino acid residues that could facilitate binding of the negatively charged phytic acid at the active
site. The enzyme did not contain bound substrate; therefore a model for substrate binding and cata-
lytic activity was proposed by the authors. In the model, the conserved histidine His 59 was in-
volved in nucleophilic attack at the 3-phosphate.
An unusual constitutive alkaline phytase that is present in lily pollen and seeds [91,92] initiates
action by first removing the 5-phosphate of phytic acid. Subsequent hydrolytic steps remove phos-
phate from the 4 and 6 position to yield Ins 1,2,3P
3
as the final product. This final product, Ins1,2,3P
3,
has been shown to inhibit iron-cata- lyzed free radical formation by chelating iron [93 –
95]. The presence of multiple phytases with differing specificity, pH optima, and biochemical
properties in wheat bran and lily pollen suggests that hydrolysis of phytic acid is under the control
of multiple phytases. We are probably entering a new phase of research which will involve under-
standing the physiological importance of the mul- tiple phytases and the biological roles of inositol
phosphates produced by them.
10. Pyrophosphorylated inositol phosphates
