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

• 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

Stages of Carbon Assimilation

Carbon Assimilation Stage 3:Regeneration of Acceptor

Transketolase Reactions

• 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

Donor1 Acceptor1 Acceptor2 Donor2

TPP

orSedoheptulose

7-phosphate

orRibose

5-phosphate

Transaldolase Reaction

• 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

Sedoheptulose 1,7-bisphosphateor

orErythrose 4-phosphate

↓↑

+

Donor Acceptor

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

bisphosphatase ↓ - Pi

sedoheptulose 7-phosphate

transketolase ↑↓ + glyceraldehyde 3-phosphate

ribose 5-phosphate + xylulose 5-phosphate

transaldolase has same ketose as substrate

transketolase has same aldose as substrate

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 ribulose 1,5-bisphosphate

3 glyceraldehyde 3-phosphate 3 ADP

3 ATP 2 Pi

Stoichiometry of Carbon Assimilation

• Assimilation of 3 carbons and 1 phosphorous per cycle

• Inorganic phosphate must be replaced for sustained ATP synthesis in chloroplast

Overall Process:

3 CO2 + 9 ATP + 6 NADPH → glyceraldehyde 3-phosphate + 9 ADP + 6 NADP+ + 8 Pi

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 enzymesRS–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

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

sulfhydryls(reduced)

disulfides(oxidized)

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

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

• Excessive amounts of triose and monosaccharide phosphates are converted to alternative forms in the light

• Liberates phosphate for ATP synthesis

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