Professional Documents
Culture Documents
Abbreviations
G6PDH glucose 6-phosphate dehydrogenase
GC
gas chromatography
MS
mass spectroscopy
NMR
nuclear magnetic resonance spectroscopy
oxPPP
oxidative pentose phosphate pathway
6PGDH 6-phosphogluconate dehydrogenase
TA
transaldolase
TK
transketolase
XPT
xylulose phosphate/phosphate translocator
Introduction
Recent years have witnessed a resurgence of interest in
the oxidative pentose phosphate pathway (oxPPP), which
Current Opinion in Plant Biology 2003, 6:236246
Figure 1
(a)
P-Gluconolactone
1 2 3 4 5 6
NADPH
6-P-Gluconate
1 2 3 4 5 6
3
Glucose 6-P
CO2
NADPH
1 2 3 4 5 6
Ribulose 5-P
Ribose 5-P
2 3 4 5 6
2 3 4 5 6
5
Fructose 6-P
6
2 3 2 4 5 6
2 3 3 4 5 6
Triose 3-P
Xylulose 5-P
4 5 6
2 3 4 5 6
Sedoheptulose-7-P
2 3 2 3 4 5 6
Erythrose-4-P
3 4 5 6
Triose 3-P
4 5 6
(b)
P-Gluconolactone
1 2 3 4 5 6
NADPH
6-P-Gluconate
1 2 3 4 5 6
3
Glucose 6-P
CO2
NADPH
1 2 3 4 5 6
Ribulose 5-P
Xylulose 5-P
8
2 3 4 5 6
2 3 4 5 6
4
Fructose 6-P
2 3 2 4 5 6
2 3 3 4 5 6
Triose 3-P
Ribose 5-P
4 5 6
2 3 4 5 6
Sedoheptulose-7-P
2 3 2 3 4 5 6
Erythrose-4-P
3 4 5 6
6
Current Opinion in Plant Biology
The oxidative pentose phosphate pathway depicted in (a) conventional and (b) alternative formulation. The enzymes ( - ) that catalyse each of the
steps are identified in Table 2. The numbers below each compound represent the fate of specific carbon atoms within the glucose 6-phosphate
that enters the pathway through the oxidative steps. Different colours are used to distinguish carbon atoms derived from ribose 5-phosphate (blue) and
xylulose 5-phosphate (green).
Table 1
Elementary flux modes of the oxidative pentose phosphate pathway.
Flux mode
Net stoichiometry
Comment
4
5
1
2
The metabolic model used for this analysis assumes that glucose 6-phosphate (Glc-6-P), pyruvate (Pyr, the end-product of glycolysis), ribose
5-phosphate (Rib-5-Pex, withdrawn for nucleotide synthesis) and CO2, together with the cofactors NADP, NADPH, NAD, NADH, ATP, ADP and
Pi, are external metabolites. The steps converting glucose 6-phosphate to ribulose 5-phosphate, converting phosphoenolpyruvate to pyruvate,
and utilising ribose 5-phosphate (Rib-5-Pex) are considered irreversible. A further six elementary flux modes occur if Rib-5-Pex is allowed to enter
the network [11].
Table 2
Summary of Arabidopsis genes likely to encode enzymes of the oxidative pentose phosphate pathway.
Enzyme
1
Glucose-6-phosphate 1-dehydrogenase
EC 1.1.1.49
6-Phosphogluconolactonase
EC 3.1.1.31
Gene number
TargetP value
Predicted location
At1g09420
At1g24280
At3g27300
At5g13110
At5g35790
At5g40760
0.895
0.926
Plastid
Plastid
0.816
0.983
Plastid
Plastid
0.964
Plastid
At1g13700
At3g49360
At5g24400
At5g24410
At5g24420
At1g64190
At3g02360
At5g41670
Ribose-5-phosphate isomerase
EC 5.3.1.6
At1g71100
At2g01290
At3g04790
At5g44520
Plastid
Plastid
0.735
0.829
Plastid
Plastid
Ribulose-5-phosphate 3-epimerase
EC 5.1.3.1
At1g63290
At3g01850
At5g61410
0.925
Plastid
Transketolase
EC 2.2.1.1
At2g45290
At3g60750
0.970
0.960
Plastid
Plastid
Transaldolase
EC 2.2.1.2
At1g12230
At5g13420
0.969
0.978
Plastid
Plastid
Glucose-6-phosphate isomerase
EC 5.3.1.9
At4g24620
At5g42740
0.949
Plastid
The identities of predicted genes within the Arabidopsis genome are based on comparison of the deduced amino-acid sequence with those of
corresponding enzymes from other organisms. The predicted subcellular location of the gene products are derived from analysis of potential
transit peptide sequences using TargetP (http://www.cbs.dtu.dk/services/TargetP/), with the exception of those for 6-phosphogluconate
dehydrogenase, which are assigned on the basis of their similarity to plastidic and cytosolic isozymes from spinach [16]. The number preceding
the name of each enzyme corresponds to the reaction(s) indicated in Figure 1.
sequences from spinach suggests that two of the Arabidopsis genes encode plastidic forms of the enzyme [16].
Thus, Arabidopsis has the genetic capacity to catalyse the
irreversible oxidative section of the oxPPP in both the
cytosol and plastids.
Similar analyses suggest that Arabidopsis contains both
cytosolic and plastidic forms of ribose-5-phosphate isomerase and ribulose-5-phosphate epimerase. For the latter enzyme, this conclusion has been corroborated by the
isolation of cDNA that encodes cytosolic and plastidic
isozymes from Arabidopsis, rice and maize [26]. In contrast, the two remaining enzymes of the non-oxidative
section of the pathway, TK and TA, may be exclusively
confined to plastids. Although two genes encode each of
these enzyme activities, all four genes contain a predicted
transit-peptide sequence, suggesting that none of the four
gene products comprising the two enzymes is cytosolic.
This implies that Arabidopsis has the genetic capacity
to convert cytosolic ribulose 5-phosphate to ribose
5-phosphate and xylulose 5-phosphate, but any further
www.current-opinion.com
Figure 2
Sucrose
Plastid
Glc-6-P
NADPH
Glc-6-P
Glc-6-P
NADPH
6-PGlcA
Pi
Pi
GPT
CO2
NADPH
Rbu-5-P
Triose-P
Pi
Triose-P
oxPPP
Rib-5-P
Pi
XPT
CO2
Nucleotides
Rib-5-P
Nucleotides
Xlu-5-P
Xlu-5-P
PEP
Ery-4-P
PEP
PPT
Pi
Pi
Shikimic acid
pathway
Cytosol
Current Opinion in Plant Biology
Exchange of oxidative pentose phosphate pathway intermediates by the plastid phosphate translocators in Arabidopsis. Glucose 6-phosphate
(Glc-6-P) can enter plastids in exchange for triose phosphate or orthophosphate (Pi) via the Glc-6-P/phosphate translocator (GPT). Exchange of
xylulose 5-phosphate (Xlu-5-P), triose phosphate (Triose-P) and Pi is catalysed by the XPT. In the absence of cytosolic transketolase and
transaldolase, this activity facilitates further metabolism (within plastids) of pentose phosphates that are generated by the oxidative reactions in the
cytosol, as well as the provision of pentose phosphates generated independently of NADPH production for nucleotide synthesis in the cytosol. The
phosphoenolpyruvate/phosphate translocator (PPT) is required for the import of phosphoenolpyruvate into plastids for the biosynthesis of aromatic
acids. The triose phosphate/phosphate translocator, which is expressed only in photosynthetic cells, is omitted for clarity.
Current Opinion in Plant Biology 2003, 6:236246
www.current-opinion.com
reduction and glutamate assimilation [1]. The involvement of the P2 form of G6PDH in the provision of
NADPH for ferredoxin-dependent reactions is suggested
by an increase in the expression of genes encoding two
Arabidopsis P2 isozymes (At1g24280 and At5g13110; see
Table 2) after transfer from a medium containing ammonium to one including nitrate [33]. Similar increases in the
expression of P2 genes upon exposure to nitrate have been
observed in the roots of tobacco and tomato [15,34].
Furthermore, recent studies on barley roots have identified a plastidic isoform of G6PDH that has properties
similar to those of P2 and that is induced in response to
exposure to ammonium or glutamate [3537]. These
observations are complemented by the demonstration that
both nitrite reduction and glutamate synthesis from glutamine and 2-oxoglutarate by plastids isolated from barley
root depend on a supply of glucose 6-phosphate. This
result confirms the ability of the P2 isozyme to support
flux through the plastidic oxPPP in spite of the high
NADPH/NADP needed to provide reductant for nitrite
reduction and glutamate synthesis [37,38].
The existence of different isozymes of plastidic G6PDH
can be rationalised, but the significance of the multiple
genes that encode other oxPPP enzymes with no obvious
regulatory properties is more difficult to explain. One
possible explanation has been suggested by studies on
transgenic tobacco plants that have decreased TK activity
[20]. The ability of darkened leaves from these plants to
stimulate flux through the oxPPP in response to wounding is compromised. More extensive analysis of photosynthetic carbon metabolism in these plants suggests that
the reduced activity of TK does not directly influence
metabolism. Rather, metabolism is perturbed by the
effects on other metabolic enzymes of changes in the
concentrations of the immediate substrates and products
of TK required to maintain flux through the reactions
catalysed by this enzyme. The relatively large decrease in
phenylpropanoid production in these transgenic plants
demonstrates the sensitivity of other processes to changes
in the levels of oxPPP intermediates. These considerations highlight the need to maintain appropriate levels of
metabolites. In this context, a plausible role for isozymes
may be to allow changes in the steady-state levels of
metabolic intermediates (owing to differences in the
relative affinities of different isozymes for substrates
and products) while maintaining the same net flux
through the pathway.
Conclusions
Considering the central importance of the oxPPP in metabolism, our understanding of its organisation and operation
in plant cells is remarkably sketchy. In part, this may be
due to the diversity of processes with which it interacts,
and the flexibility required to meet the varying demands of
such processes for reductant and biosynthetic precursors.
However, prospects for improving our appreciation of the
oxPPP are good. As more genomes are sequenced, we will
develop a better understanding of the genetic capacity of
plant systems to catalyse the reactions involved in the
oxPPP, and have access to a greater range of cognate genes
for comparative analysis [54]. Furthermore, the increasing
availability of whole-genome transcript profiles under
different physiological and developmental conditions will
allow us to determine the relationships between the
expression of specific oxPPP isozymes, and to establish
the contribution of such isozymes to particular physiological processes [55,56]. Hopefully, such comparisons can
be complemented by the development of equally robust
technologies for monitoring changes in the profiles of
a broad range of metabolites. This will enable a full
assessment of the possibly wide-ranging metabolic consequences of perturbations in the expression of specific
oxPPP genes [57]. Finally, conceptual advances in the
way in which groups of enzyme-catalysed reactions are
www.current-opinion.com
Acknowledgements
Research in the authors laboratories is funded by the Biotechnology and
Biological Sciences Research Council (UK) and Deutsche
Forschungsgemeinschaft (Germany).
2.
3.
4.
5.
6.
7. van Winden WA, Verheijen PJT, Heijnen JJ: Possible pitfalls of flux
calculations based on 13 C-labeling. Metab Eng 2001, 3:151-162.
This paper provides a timely reminder of potential problems in the use of
steady-state labelling strategies for flux determination. The authors show
that exchange reactions catalysed by transketolase and transaldolase
that have no net stoichiometric effect (and thus are often ignored)
influence the labelling pattern of metabolites. Failure to consider these
exchange reactions can lead to erroneous estimates of flux throughout
the metabolic network, even though the calculated flux values might
provide a good fit to the observed labelling pattern.
8.
9.
www.current-opinion.com