Carbohydrate Biosynthesis in Plants
CH353 January 15, 2008
Overview of Plant Metabolism
Overview of Carbon Assimilation
Occurs in Chloroplasts
Stage 1: Fixation
•
1 step – RUBISCO
unique to plants
Stage 2: Reduction
•
3 steps – analogous
to gluconeogenesis
(uses NADPH)
Stage 3: Regeneration
•
9 steps; 7 enzymes
analogous to pentose
phosphate pathway
Stages of Carbon Assimilation
• Stage 1: Fixation
Rubisco
ribulose 1,5-bisphosphate + CO2 → 2 3-phosphoglycerate
• Stage 2: Reduction
3-phosphoglycerate kinase
3-phosphoglycerate + ATP → 1,3-bisphosphoglycerate + ADP
glyceraldehyde 3-phosphate dehydrogenase
1,3-bisphosphoglycerate + NADPH →
glyceraldehyde 3-phosphate + NADP+ + Pi
triose phosphate isomerase
glyceraldehyde 3-phosphate ↔ dihydroxyacetone phosphate
Carbon Assimilation Stage 3:
Regeneration of Acceptor
Transketolase Reactions
Donor1
Acceptor1
Acceptor2
Donor2
TPP
or
Sedoheptulose
7-phosphate
or
Ribose
5-phosphate
• Transketolases transfer “active aldehyde” from a ketose (donor) to
an aldose (acceptor) with cofactor thiamine pyrophosphate (TPP)
• Transketolase reactions for carbon assimilation in chloroplast are
identical to those for pentose phosphate pathway in cytosol
Transaldolase Reaction
Sedoheptulose 1,7-bisphosphate
or
Donor
↓↑
Acceptor
+
or
Erythrose 4-phosphate
• Transaldolases transfer dihydroxyacetone phosphate (donor) to an
aldose (acceptor) forming an aldol
condensation adduct
• Involves Schiff base enzyme bound
intermediate
• Transaldolase reaction (pictured) is
identical to aldolase reaction in
glycolysis/gluconeogenesis; other is
unique to carbon assimilation
• Donor: dihydroxyacetone phosphate
• Acceptors: erythrose 4-phosphate
and glyceraldehyde 3-phosphate
Stage 3: Regeneration of Acceptor
glyceraldehyde 3-phosphate
transaldolase
↑↓ + dihydroxyacetone phosphate
fructose 1,6-bisphosphate
bisphosphatase
↓ - Pi
fructose 6-phosphate
transketolase
↑↓ + glyceraldehyde 3-phosphate
transaldolase
has same ketose
as substrate
erythrose 4-phosphate + xylulose 5-phosphate
transaldolase
↑↓ + dihydroxyacetone phosphate
sedoheptulose 1,7-bisphosphate
bisphosphatase
↓ - Pi
transketolase
has same aldose
as substrate
sedoheptulose 7-phosphate
transketolase
↑↓ + glyceraldehyde 3-phosphate
ribose 5-phosphate + xylulose 5-phosphate
bisphosphatases
make process
irreversible
Stage 3: Regeneration of Acceptor
2 xylulose 5-phosphate
1 ribose 5-phosphate
ribulose 5-phosphate epimerase ↑↓
↑↓ ribose 5-phosphate isomerase
3 ribulose 5-phosphate
ribulose 5-phosphate kinase
↓
+ 3 ATP → 3 ADP
3 ribulose 1,5-bisphosphate
Stage 3 Net:
Input: 15 C
Output: 15 C
2 dihydroxyacetone phosphate
3 glyceraldehyde 3-phosphate
3 ATP
3 ribulose 1,5-bisphosphate
3 ADP
2 Pi
Stoichiometry of Carbon Assimilation
Overall Process:
3 CO2 + 9 ATP + 6 NADPH → glyceraldehyde 3-phosphate + 9 ADP + 6 NADP+ + 8 Pi
•
•
Assimilation of 3 carbons
and 1 phosphorous per cycle
Inorganic phosphate must be
replaced for sustained ATP
synthesis in chloroplast
Phosphate–Triose Phosphate Antiporter
• Exchanges dihydroxyacetone phosphate or 3-phosphoglycerate
for phosphate
• In light: triose phosphate transported to cytosol with antiport of
phosphate to chloroplast stroma
• Phosphate is released in cytosol with sucrose biosynthesis
ATP and Reducing Equivalents Exchange
•
•
•
•
Exchange of ATP and
reducing equivalents
mediated by antiporter
only 3-phosphoglycerate
or dihydroxyacetone
phosphate transported
ATP and NADPH used
on stromal side and ATP
and NADH generated on
cytosolic side
no net flux of phosphate
or triose phosphate
Regulation of Enzymes
• Rubisco
– Rubisco activase removes substrate from inactive enzyme
(ATP hydrolyzed)
– Carbamoylation of active site lysine (CO2 + Mg+2)
– Nocturnal inhibitor binds
• Photosynthetic environment in chloroplast stroma
↑ NADPH
↑ pH
↑ Mg2+
– Conditions stimulate enzyme activity
– Rubisco activation (carbamoyllysine formation) is faster
– Fructose 1,6-bisphosphatase activity ↑ 100x with illumination
• Reduction of enzymes
RS–SR’ → RSH + HSR’
Regulation of Enzymes
• Photosynthetic environment in chloroplast stroma
↑ NADPH
↑ pH ↑ Mg2+
Effect of pH and [Mg2+] on activity
of fructose 1,6-bisphosphatase
Regulation of Enzymes
sulfhydryls
(reduced)
disulfides
(oxidized)
Activated by Reduction of Disulfides
•
•
•
•
glyceraldehyde 3-phosphate dehydrogenase
fructose 1,6-bisphosphatase
sedoheptulose 1,7-bisphosphatase
ribulose 5-phosphate kinase
Inactivated by Reduction:
•
glucose 6-phosphate dehydrogenase
Rubisco Oxygenase Activity
• Rubisco accepts both CO2 and
O2 as substrates
• Incorporation of O2 into ribulose
1,5-bisphosphate produces:
– 3-phosphoglycerate
– 2-phosphoglycolate
• No fixation of CO2
• Requires 2-phosphoglycolate
salvage
Glycolate Pathway
• Salvage of 2-phosphoglycolate
• Involves metabolite transport and
enzymes in chloroplast,
peroxisome and mitochondrion
• Glycine decarboxylase is key
enzyme
• Process consumes O2 and
evolves CO2 “Photorespiration”
• Wastes energy and fixed carbon
and nitrogen
C4 Pathway
• Rubisco oxygenase activity
favored by high temperature/low
moisture environments
• C4 plants separate fixation of
HCO3- and CO2 in different but
metabolically-linked cells
• Requires more energy (2 ATP’s)
but avoids wasteful oxygenase
reaction
• CAM plants temporally separate
2 fixations (store malate at night)
Starch and Sucrose Biosynthesis
• Excessive amounts of triose and monosaccharide phosphates
are converted to alternative forms in the light
• Liberates phosphate for ATP synthesis
Starch Biosynthesis
• Carbohydrate storage
• Occurs in plastids
• ADP-glucose substrate
• Adds to reducing end (unlike
glycogen synthesis)
• α(1→4) glucose (amylose)
with α(1→6) branches
(amylopectin)
Sucrose Biosynthesis
• Carbohydrate transport
• Occurs in cytoplasm
• Fructose 6-phosphate &
UDP-glucose
• Joins reducing (anomeric)
hydroxyls
• Glucose(α1↔β2)Fructose
Cellulose Biosynthesis
•
•
•
•
Cell wall structure
Occurs in cytoplasm and at plasma membrane
Lipid-linked carrier and membrane protein complex
UDP-glucose is generated from sucrose and UDP by
sucrose synthase
• UDP-glucose is substrate for cellulose synthase;
adds glucose monomers to non-reducing end
• Cellulose is β(1→4) linked glucose
Regulation of Sucrose Biosynthesis
• Need phosphate for ATP synthesis and
triose phosphate for carbon fixation
• Fructose 2,6-bisphosphate (F2,6BP)
activates pyrophosphate-dependent
phosphofructokinase-1 (PP-PFK-1)
and inhibits fructose bisphosphatase-1
(FBPase-1)
• Its synthesis by phosphofructokinase-2
is inhibited by triose phosphates (light)
and activated by phosphate (dark)
• In dark: ↑ Pi, ↑ F2,6BP, ↑ F1,6BP →
glycolysis
• In light: ↑ triose phosphates, ↓ F2,6BP,
↑ F6P → sucrose biosynthesis
Regulation of Sucrose Biosynthesis
• Sucrose 6-phosphate synthase
(SPS) is partially inactivated by
phosphorylation by SPS kinase
• In light: glucose 6-phosphate
(high gluconeogenesis) directly
stimulates SPS and inhibits
SPS kinase activating SPS
(sucrose biosynthesis)
• In dark: phosphate directly
inhibits SPS and inhibits SPS
phosphatase inactivating SPS
(no sucrose biosynthesis)
Regulation of Starch Biosynthesis
ADP-glucose pyrophosphorylase synthesizes starch precursor
• inhibited by high [Pi] accumulating in the dark (ATP hydrolysis)
• activated by high [3-phosphoglycerate] accumulating in the light
(carbon assimilation; diminished sucrose biosynthesis)
Gluconeogenesis from Fats
• Germinating seeds convert stored fats
into sucrose
• β-oxidation (glyoxysome) fatty acid →
acetyl-CoA
• glyoxylate cycle converts 2 acetyl-CoA →
succinate
• mitochondrial citric acid cycle &
cytoplasmic gluconeogenesis converts
succinate → hexoses