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
Recently, inositol phosphates containing one or more pyrophosphate groups, have been detected in
slime mold and mammalian cells [78,79]. Com- pounds with a pyrophosphate group at the 1 or 5
position of phytic acid pyrophosphoinositol pen- takisphosphate, PP-InsP
5
or two pyrophosphate groups tentatively placed at 1 and 5 or at 5 and 6
positions bispyrophosphoinositol
tetrakisphos- phate, [PP]
2
-InsP
4
have been identified Fig. 10. A phytic acid-kinase, which can convert the phos-
Fig. 10. Pyrophosphorylated inositol polyphosphates. A 5-PP-InsP
5
and B 1,5-[PP]
2
-InsP
4
.
phate group at the 5 position to a high energy pyrophosphate group, and a PP-InsP
5
kinase, which can convert a second phosphate to a py-
rophosphate group, have been isolated [96]. These high-energy pyrophosphates are not metabolically
static. They exhibit rapid turnover through the action of phosphatases and kinases. Estimates are
that 30 – 50 of the cellular phytic acid cycles through these pyrophosphorylated derivatives ev-
ery hour [97]. The potential involvement of py- rophosphorylated inositol phosphates in signal
transduction and calcium metabolism is suggested by the observation that in a muscle cell line, the
concentration
of [PP]
2
-InsP
4
declined rapidly
70 by activation of the cAMP-dependent path- way and that sigargin, which increases cytoplasmic
calcium by depleting calcium stores in endoplas- mic reticulum, reduced the concentration of [PP]-
InsP
5
and [PP]
2
-InsP
4
by 50 reviewed in Shears [79]. Such tantalizing bits of information indicate
that these metabolically-active, high energy phos- phates could play multiple roles in phosphate and
calcium metabolism and suggest that further work is necessary for a better understanding of their
cellular roles.
11. Phosphatidylinositol and its polyphosphates
Due to the critical role that phosphoinositides play in signal transduction, this area of inositol
chemistry has been actively investigated and its current status in both animals and plants has been
comprehensively reviewed [98]. Phosphatidylinosi- tol PtdIns and its phosphorylated derivatives,
PtdIns-4-phosphate PtdInsP and PtdIns-4,5-bis- phosphate PtdInsP
2
, are present in plant tissues [98] but their functions as extracellular signals
remains an equivocal issue [99]. As used in this review, the abbreviation PtdIns refers to PtdIns
with a MI head group. If the head group is other than MI, the isomeric inositol will be specified. In
plants, the biosynthesis of PtdIns via the CDP-dia- cylglycerol pathway is well established [98] Fig.
11, as is subsequent phosphorylation of PtdIns by specific kinases to PtdInsP and PtdInsP
2
. More recently,
the presence
of the
3-substituted polyphosphoinositides,
PtdIns3P
1
and Pt-
dIns3,4P
2
, in plant cells has been documented [98].
Besides variability in the position and number of phosphate groups on the inositol ring, another
source of structural heterogeneity in phosphoinosi- tides has been the presence of isomeric inositols.
Although MI is the predominant form in the majority of inositol-containing compounds, the
presence of scyllo-inositol-containing PtdIns des- ignated PtdscylloI has been found in plant cells
and chiro-inositol-containing PtdIns designated PtdchiroI in animal cells. Isomeric phosphoinosi-
tides could be synthesized by unique CDP- DG:inositol 3-phosphatidyltransferase, also called
PtdIns synthase, such as CDP-DG:scyllo-inositol 3-phosphatidyl transferase Fig. 11, step 4 or by
exchange of the head group, MI for scyllo-inositol by PtdIns:scylloI phosphatidyltransferase, also
called the exchange enzyme Fig. 11, step 5. At present there is evidence for both pathways. In
vitro studies in barley aleurone cells have indicated the presence of CDP-DG:scyllo-inositol trans-
ferase activity. Taken together, in vivo and in vitro studies suggest that PtdscylloI is biosynthesized by
the CDP-DG:scyllo-inositol transferase pathway [100]. Investigations in mammalian cells [101] and
soybean seedlings [102] suggested that PtdchiroI is synthesized via PtdIns either by the operation of a
head group exchange enzyme or an epimerase. In this context, it is important to note the cloning
studies in mammalian cells which indicated that a single polypeptide exhibited both the synthase and
CMP-dependent exchange activities [103]. There- fore, further work is required to demonstrate
clearly that head group transfer activity is not involved in the biosynthesis of PtdscylloI in barley
aleurone cells.
12. Glycosyl-phosphatidylinositol and Glycosyl-inositolphosphorylceramide
