Fig. 2. myo-Inositol.
If one takes biosynthesis as a basis of assign- ment and traces the origin from
D
-glucose 6-phos- phate,
then clockwise
assignment preserves
biosynthetic relatedness. Unhappily, recent ad- vances involving key roles for MI polyphosphates
involved in signal transduction where addition or loss of a single phosphate may alter assignment
from
D
- to
L
- or vice-versa have created confusion for those unfamiliar with rules of cyclitol nomen-
clature. The upshot is a tentative agreement by the International Union of Biochemistry to relax rules
of nomenclature so that
1L
-MI-1-P, the product of myo-inositol-1-phosphate synthase, may be desig-
nated
1L
-MI1P
1
,
1D
-MI3P
1
, or simply, Ins3P
1
where the symbol Ins signifies MI with counter- clockwise
numbering from
1D
. Thus,
1D
- MI1,4,5P
3
, an important physiological signal generated during phosphatidylinositol-4,5-bisphos-
phate metabolism, becomes simply Ins1,4,5P
3
. More detailed discussion of the stereochemistry of
MI and its phosphate esters is found in [14] and on the Internet at http:www.chem.qmw.ac.uk
iubmbnomenclature.
3. MI biosynthesis
3
.
1
. E6idence for cyclization of
D
-glucose to MI Although the
D
-gluco configuration inherent in MI was recognized by Maquenne as early as 1887
and the proposition that
D
-glucose-6-phosphate cyclized to form Ins3P
1
enzymatically was ad- vanced by H.O.L. Fischer in 1945, unequivocal
evidence for conservation of the 6-carbon chain of
D
-glucose during cyclization to MI did not appear until 1962 [20,21].
The experimental approach involved recovery of labeled MI from a
D
-[1-
14
C]glucose-labeled parsley leaf followed by administration of this labeled MI
to detached immature strawberry fruits where it was utilized as a carbon source for pectin biosyn-
thesis [20,22]. Carbon-14 was recovered in
D
-galac- turonosyl and
L
-arabinosyl residues of pectin which upon radioanalysis revealed 79 of the
label in carbon 1. Distribution of
14
C in sucrose- derived
D
-glucose, pectin-derived
D
-galacturonate and
L
-ascorbic acid from the parsley leaf also had \
80 of their
14
C in carbon 1. In other words, about 80 of the
14
C in these products of
D
-1-
14
Cglucose metabolism remained at the original
Fig. 3. Conventions for numbering substituents in myo-inosi- tol.
Biosynthesis of phytic acid MI-P
6
and phytic acid pyrophosphates [14 – 16].
Metabolic recycling of products of phytic acid hydrolysis during phytase-mediated phytic acid
dephosphorylation [14 – 16].
Biosynthesis of phosphatidylinositol, its poly- phosphates, and precursors of MI polyphos-
phate-specific signal transduction [1,2,14,17].
Glycosylated-phosphatidylinositol and glycosy- lated-inositolphosphorylceramide [18,19].
2. Nomenclature
The nomenclature of inositols has been an on- going source of confusion and conflict for decades.
MI is a meso compound with a plane of symmetry that rotates the structure about C2 and C5 as fixed
positions Fig. 2. The remaining four carbon atoms consist of two prochiral pairs, C1 = C3 and
C4 = C6. If the carbon ring is numbered clock- wise, as shown by numbers inside the ring, assign-
ment of a single substituent on carbon 1 is
1L
. Conversely, if the carbon ring is numbered coun-
terclockwise as shown by numbers external to the ring, assignment is
1D
.
1L
-MI-1-P, the product of
1L
-myo-inositol-1-phosphate synthase
E.C.5.5. 1.4, is a good example of the dilemma this choice
creates Fig. 3.
site of labeling. Some redistribution of
14
C be- tween terminal carbons is normal during triose
hexose phosphate metabolism. This analytical approach provided profound in-
sight into three aspects of MI metabolism, namely MI biosynthesis, the MI oxidation pathway, and
phytic acid biosynthesis. It provided evidence for cyclization of the carbon chain of
D
-glucose to form MI. It revealed an alternative biosynthetic
pathway to uronic acid and pentose constituents of plant cell wall polysaccharides quite indepen-
dent of the one involving UDP-
D
-glucose dehy- drogenase. Finally, it supplied stereochemical
evidence for the putative initial phosphorylated intermediate leading to phytic acid, a major form
of phosphate storage in plants.
3
.
2
. Mechanism of MI biosynthesis Biosynthetic conversion of
D
-glucose to free MI involves three enzymatic steps Fig. 4. Step B,
cyclization of
D
-glucose-6-P to Ins3P
1
, is the first committed step in MI biosynthesis. Step C, loss of
phosphate, releases free MI. Overall, this scheme constitutes the sole pathway of MI biosynthesis in
cyanobacteria, algae, fungi, plants, and animals and occupies a central role in their cellular
metabolism.
Cyclization of
D
-glucose-6-phosphate to
Ins3P
1
is irreversible. This process also highlights a dilemma in nomenclature since
D
-glucose is numbered clockwise about the pyranose ring while
conventional numbering of MI inverts the num- bering of its inherent
D
-gluco configuration. Ins3P
1
synthase appears to be a highly con- served enzyme [23]. Functionally, this conversion
of
D
-glucose 6-P to Ins3P
1
involves three sub- steps Fig. 5:
NAD
+
-coupled oxidation of carbon 5 of
D
- glucose-6-P.
Aldol condensation between carbon 1 and car- bon
6 of
5-keto-
D
-glucose-6-P
D
-xylo-5- hexulose-6-P.
NADH-catalyzed reduction of 2-myo-inosose- 1-P
D
-2,4,63,5-pentahydroxy-cyclohexane-2-P to yield Ins3P
1
. Specific points of interest regarding Ins3P
1
syn- thase include:
Preference for the b-anomeric form of glucose- 6-P.
Enzyme-bound NAD removable by charcoal treatment to generate an inactive apo-enzyme.
NAD
+
-catalyzed oxidation at carbon 5 of glu- cose-6-P substep 1 to yield an enzyme-bound
5-keto-glucose-6-P with hydride ion transfer from glucose-6-P to the pro-S position of car-
bon 4 on the nicotinamide moiety of NAD
+
Kinetic studies indicate a sequential reaction with NAD
+
adding first. There is a distinct isotope effect in removal of hydrogen from
carbon 5 of glucose-6-P..
Base-catalyzed cyclization substep 2. — Oxy- gen at carbon 5 is retained. The pro-R hydro-
gen is removed from carbon 6 while the pro-S hydrogen is retained. The second intermediate
within brackets is 2-myo-inosose-1-P
D
-2,4,6 3,5-pentahydroxycyclohexanone-2-P. For addi-
tional details, see [24].
Fig. 4. Conversion of
D
-glucose to MI: A Hexokinase, EC 2.7.1.1; B Ins3P
1
synthase, EC 5.5.1.4; C MI monophosphatase, EC 3.1.3.25.
Fig. 5. Enzymatic mechanism of
1L
-MI-1-phosphate synthase a.k.a. Ins3P
1
synthase.
Stereospecific oxidation
of enzyme-bound
NADH substep 3 including transfer of its pro-S hydride ion to the si face of the carbonyl
group to generate Ins3P
1
.
An undisturbed phosphate – carbon bond. Hydrolysis
of Ins3P
1
by a
specific MI
monophosphatase [4] completes overall conversion of
D
-glucose to free MI.
3
.
3
. Biochemical and physiological aspects of Ins
3
P
1
synthase The structural gene INO1 for Ins3P
1
synthase was first isolated from the yeast Saccharomyces
cere6isiae by Donahue and Henry [25]. Subsequent studies involving ino
1
mutations in MI auxotrophs provided insight into regulatory control processes
[3,26]. Transcripts with homology to this gene have been obtained from several plant sources and
are summarized in these cited references [3,23]. The first such plant gene for Ins3P
1
synthase to be characterized was tur1, a cDNA from the duck-
weed, Spirodela polyrrhiza, which was rapidly and spatially up-regulated during an ABA-induced
morphogenic response [27]. This effect was local- ized to stolon tissue that connects the developing
turion to the node of the mother frond. The authors considered several possible scenarios in
which MI synthesis might play a role. These in- cluded phytic acid accumulation, lipid synthesis,
an ABA signal transducing mechanism involving phosophoinositide metabolites, altered flux of MI
metabolites into the cell wall andor an auxin- linked cell elongation involving auxin conjugates,
and induced response to stress involving methyl ethers of inositol. Of these possibilities, an effect
on the nature of cell wall structures appeared most interesting. Since S. polyrrhiza was not yet trans-
formable at the time of these studies, Smart and Flores [28] generated transgenic Arabidopis plants
over-expressing Ins3P
1
synthase TUR1 cDNA from S. polyrrhiza and found these plants to con-
tain elevated Ins3P
1
synthase activity with con- comitant four-fold increase in endogenous MI.
Comparison of transgenic to wild-type plants re- vealed no significant differences in whole plant
growth habit, expansion growth, germination rate, flowering time, stem thickness, in vitro root
growth and germinationsurvival on high salt or low temperature regimes. A four-fold increase in
endogenous MI may have been insufficient to trigger gross differences in growth or development
although compositional differences have been ob- served in lily pollen germinated in media that was
supplemented with MI ranging from 0.3 to 2.8 mM [29].
In algae and plants, both cytosolic and chloro- plastic forms of Ins3P
1
synthase have been iso- lated
and characterized
[30]. Although
the biochemical and kinetic parameters of these two
forms do not differ significantly between each other or from other cytosolic Ins3P
1
synthases previously described [3], the native cytosolic form
is homotrimeric while the native chloroplastic form is homotetrameric. Interestingly, a cyanobac-
terium, Spirulina platensis, included in the cited study, contained only one cytosolic homote-
trameric form as anticipated by the endosymbiont theory for a cyanobacterial origin of plastids
[3,31,32].
Analysis of an INO1-like transcript termed INPS1 by the authors [23] from salt-stressed
Mesembryanthemum crystallinum ice plant re- vealed a diurnal fluctuating increase in mRNA
during the light period that could be coordinated with the gene encoding MI-O-methyltransferase,
an enzyme methylating MI to
D
-ononitol which is epimerized to
D
-pinitol.
D
-Pinitol accumulates in salt-stressed M. crystallinum plants and is consid-
ered to be the principal osmoregulator. Compara- ble
experiments with
Arabidopsis thaliana
transcripts failed to produce this effect and the authors conclude that there is probably no stress-
mediated induction of Inps
1
mRNA in Arabidop- sis, an observation similar to that made by Smart
and Flores [28]. Salt-tolerant varieties of rice grown in a NaCl environment exhibited a photore-
sponsive enhancement of chloroplast and cytosolic Ins3P
1
synthase activity [33]. The authors specu- late on the possible role of free MI as an osmolyte
in the chloroplast through coordinate activation andor induced expression of Ins3P
1
synthase and MI monophosphatase.
Keller et al. [34] obtained a full-length cDNA from
potato epidermal
tissue that
encoded Ins3P
1
synthase termed StIPS-
1
. RNA blot analysis revealed the highest StIPS-
1
transcript levels in photosynthetic tissues but much lower
levels in roots and tubers. Light greatly elevated StIPS-
1
transcript levels but drought stress had no effect. When antisense StIPS-
1
transformants were tested, it was found that their leaves had
strongly reduced levels of MI, galactinol, and raffinose. These plants also showed distinct mor-
phological aberrations including decreased overall tuber yield. These findings highlight broad bio-
chemical and physiological effects brought on by altering Ins3P
1
synthase activity in potato and possibly most other plant species.
Quite recently, Yoshida and co-workers [35] isolated a cDNA clone, pRINO1, from rice Oryza
sati6a L. callus suspension cultures that is highly homologous to Ins3P
1
synthase from yeast and plants. Its transcript appears in the apical region
of globular-stage embryos 2 days after anthesis and strong signals were detected in the scutellum
and aleurone layer after 4 days. Phytate-contain- ing particles or globoids appeared in the same
tissues at 4 days, coinciding with the RINO1 transcript. This study demonstrates that Ins3P
1
synthase is probably the first committed step in phytic acid biosynthesis although a complemen-
tary process involving salvage of MI by MI kinase remains untested.
In studies just reviewed, genes encoding Ins3P
1
synthase are variously identified as INO1 Saccha- romyces cere6isiae, TUR1 Spirodela polyrrhiza,
StIPS-1 Solanum tubersum, INPS1 Mesem- bryanthemum crystallinum, pRINO1 Oryza sa-
ti6a, etc. Deduced amino acid sequences obtained from these plant-derived sources are quite similar.
In the interest of consistency, a common term like INPS1 as proposed by Ishitani et al. [23] seems
desirable.
3
.
4
. Concerning free MI Dephosphorylation of Ins3P
1
constitutes the sole de novo route to free MI in plants. All other
sources derive from salvage mechanisms involving recovery of free MI from other metabolic MI-con-
taining products. Free MI is generally regarded as a ubiquitous constituent of plant tissues and in
some species, notably Actinidia arguta, kiwifruit, MI is the major ‘sugar’ constituent 60 – 65 dur-
ing the first 20 – 30 days after anthesis [36]. Stress- related aspects of MI accumulation have been
noted repeatedly [1,2,6,23,37,38] but specific bio- chemical and molecular details are needed. In fact,
accumulation of free MI may be a more universal phenomenon in life forms than generally realized.
In overwintering ladybird beetles Creatomegilla undecimnotata, free MI, functioning as a possible
cryoprotectant, increases more than four-fold from 2.5 to 11 mgmg wet weight during winter
months [39]. A relatively specific alkaline, magnesium-depen-
dent phosphatase MI monophosphatase, EC 3.1.3.25 from lily pollen hydrolyzes Ins3P
1
, its enantiomer Ins1P
1
, and at a somewhat lower rate, Ins2P
1
, to free MI [40,41]. Animal tissues contain a similar MI monophosphatase [42] but
Ins2P
1
which is substituted in the axial position is not a substrate although it does act as a compet-
itive inhibitor in the case of bovine brain enzyme. There is need to revisit this matter of substrate
specificity since most plant studies are dated in this regard.
Gillaspy et al. [4] cloned three MI monophos- phatase activities from tomato LeIMP. All iso-
forms were
lithium ion-sensitive
over a
concentration range similar to that exhibited by human MI monophosphatase. When labeled anti-
sense RNA probes were used to follow mRNA accumulation of these isoforms at different devel-
opmental stages and in different organs, LeIMP
1
mRNA accumulation was greatest in light-grown seedlings, flowers, young and mature green fruit
decreasing as fruit proceeded to the breaker stage, and callus tissue. LeIMP
3
mRNA was detected in the same tissues and had a much
higher response in the shoot apex. LeIMP
2
mRNA levels were significantly lower than the other two isoforms. Consideration of apparent
differences in the expression patterns of these three isoforms, prompted the suggestion that their activ-
ities functioned within different cell types or spa- tially distinct cellular compartments. Evidence to
support spatially regulated expression has since been presented [43]. Given the diverse demands for
free MI as outlined in Fig. 1 and the ancestral history of plant organelles, such a suggestion is
quite possible. Clearly, there is need for more information on localization of MI monophos-
phatase within the plant cell.
Inhibition of MI monophosphatase by lithium ions is especially noteworthy. At 0.1 mM Li
+
, bacterial protein extracts, each expressing one of
the LeIMP gene products, had less than 20 of maximal activity for removing phosphate from
[
14
C]MI-1-P [4]. Although phytotoxicity of Li
+
has been studied for decades, efforts to pinpoint its
specific targets in plant cells have yielded only meager results [44]. Beyond its inhibitory effect on
Fig. 6. Cell wall polysaccharide biogenesis via the myo-inositol oxidation pathway bold font and the sugar nucleotide oxidation pathway.
MI monophosphatase, little is known regarding its impact on availability of free MI for numer-
ous biosynthetic and regulatory requirements Fig. 1. Lithium ion delayed initiation of DNA
synthesis and cell division when introduced into synchronized Catharanthus roseus cell cultures, a
condition largely prevented when MI was in- cluded in the medium. Preparations of MI
monophosphatase from C. roseus were inhibited 80 by 10 mM Li
+
[45]. Use of Li
+
inhibition as a tool for studying modulation of free cellular
MI appears to be a viable option, one which may also alter other metabolic pathways con-
nected to a demand for free MI such as its role as substrate for MI kinase EC 2.7.1.64. Curi-
ously, little attention has been given to this latter enzyme which is present in plants, animals and
microorganisms [46]. Its product, Ins3P
1
, has the same configurational structure as that pro-
duced by Ins3P
1
synthase [5]. While one might regard this recycling of MI back into a pool of
Ins3P
1
as a salvage mechanism, it fails to take into consideration any localization of these en-
zymic activities or temporal demands during de- velopment. Together, Ins3P
1
synthase and MI kinase constitute ways in which Ins3P
1
is formed from
D
-glucose-6-P or free MI in plants. The former enzyme is biosynthetic while the lat-
ter must rely on sources that generate free MI from MI monophosphatase or other MI-conju-
gated forms. Unresolved are temporal and spa- tial patterns of synthase and kinase during
growth and development.
4. myo-Inositol oxidation pathway
