1. Carbohydrate Biosynthesis in Plants
CH353 January 15, 2008

2. Overview of Plant Metabolism

3. 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

4. Stages of Carbon Assimilation
Stage 1: Fixation
Rubisco
ribulose 1,5-bisphosphate + CO2 → 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

5. Carbon Assimilation Stage 3: Regeneration of Acceptor

6. Transketolase Reactions
Donor Acceptor 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

7. Transaldolase Reaction
Sedoheptulose 1,7-bisphosphate or
Transaldolases transfer dihydroxy-acetone 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 ↓↑ +
Donor
Acceptor or
Erythrose 4-phosphate

8. Stage 3: Regeneration of Acceptor
glyceraldehyde 3-phosphate
transaldolase ↑↓ + dihydroxyacetone phosphate
fructose 1,6-bisphosphate
bisphosphatase ↓ - Pi
fructose 6-phosphate
transketolase ↑↓ + glyceraldehyde 3-phosphate
erythrose 4-phosphate + xylulose 5-phosphate
transaldolase ↑↓ + dihydroxyacetone phosphate
sedoheptulose 1,7-bisphosphate
sedoheptulose 7-phosphate
ribose 5-phosphate xylulose 5-phosphate
transaldolase has same ketose as substrate
transketolase has same aldose as substrate
bisphosphatases make process irreversible

9. Stage 3: Regeneration of Acceptor
2 xylulose 5-phosphate 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 ribulose 1,5-bisphosphate
3 glyceraldehyde 3-phosphate ADP
3 ATP Pi

10. 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

11. 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

12. 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

13. 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’

14. Regulation of Enzymes Photosynthetic environment in chloroplast stroma
↑ NADPH ↑ pH ↑ Mg2+
Effect of pH and [Mg2+] on activity of fructose 1,6-bisphosphatase

15. Regulation of Enzymes Activated by Reduction of Disulfides
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

16. 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

17. 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

18. 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)

19. 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

20. 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

21. 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

22. 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)

23. 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)

24. 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