Lecture #20 - Isoprenoid Metabolism (IN PROGRESS)

Isoprenoids

Largest and most chemically-diverse class of natural compounds (~50,000 in plants).

Isoprenoids - Functions

1. Alkoloids

2. Bile salts

3. Chlorophyll

4. Carotenoids

5. Coenzyme Q

6. Cholesterol

7. Dolichol

8. Farnesylation

9. Fat-soluble vitamins

10. Haem A

11. Hormones

12. Juvenile hormones

13. Peptidoglycan

14. Plastoquinone

15. Sterols

16. Terpenes

17. Taxol

18. Natural rubber

Isoprenoids - Synthesis (Overview)

Isoprenoids are synthesized from 2 isomeric hemiterpene precursors:

1. Isopentenyl Disposphate (IPP)

2. Dimethylallyl Diphosphate (DMAPP)

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Isoprenoids are synthesized from 2 distinct pathways:

1. Mevalonic Acid (MVA) Pathway – Cytosolic pathway (plants and animals).

2. Methylerythritol Phosphate (MEP)/Deoxyxylulose (DXP) Pathway – Plastidial pathway (Plants, protozoa, and bacteria).

Isoprenoids - Synthesis (MVA Pathway)

1. Condensation: 2 x Acetyl-CoA → Acetoacetl-CoA + CoASH (via Acetyl-CoA Thiolase 2 (AACT2))

NOTE: AACT identified by functional complementation of yeast Δerg10 mutant, AACT2 identified by biochemical characterization.

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2. Aldol Condensation: Acetoacetyl-CoA → 3-Hydroxyl-3-Methylglutaryl-CoA (HMG-CoA) + CoASH (via HMG-CoA Synthase (HMGS)).

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3. NADH-Dependant Reduction: HMG-CoA + 2 x NADPH → MVA + 2 x NADP+ (via HMG-CoA Reductase (HMGR)).

NOTE:

• HMGR Characteristics:

  • Key enzyme: one of the most regulated enzymes (feedback, transcription, translation, phosphorylation, degradation).

  • Inhibited by mevinolin (lovastrin) via competitive inhibition: binds to active site of HMGR, inhibiting MVA production and downstream products.

• HMGR Structure:

  • Conserved membrane anchor.

  • Highly-variable hydrophilic N-terminal sequence.

    • Differences in sequence and degree of glycosylation affects targeting and activity.

  • Highly-variable linker sequence.

  • Highly-conserved, cytosol-exposed C-terminal catalytic domain.

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4. ATP-Dependant Phosphorylation: MVA + ATP → Mevalonic Acid-5-Phosphate (MVAP) + ADP (via Mevalonate Kinase (MVK)).

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5. ATP-Dependant Phosphorylation: MVAP + ATP → Mevalonic Acid-5-Diphosphate/Mevalonate Diphosphate (MVAPP) + ADP (via Phosphomevalonate Kinase (PMK)).

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6. ATP-Dependant Decarboxylation: MVAP + ATP → Isopentenyl Pyrophosphate (IPP) + Pi + CO2 + H2O (via 5-Diphosphomevalonate Decarboxylase (PMD)).

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7. Isomerization: IPP → Dimethylallyl Diphosphate (DMAPP) (via IPP Isomerase (IDI)).

Isoprenoids - Synthesis (MEP Pathway)

1. 
   

PLANTS:

A) Transketolase-Like Decarboxylation: Pyruvate + Glyceraldehyde-3-Phosphate (G3P) → 1-Deoxy-Xylulose-5-Phosphate (DXP) + CO2 (via DXP Synthase (DXS)).

BACTERIA:

A) Transketolase-Like Decarboxylation: Pyruvate + Thiamine Pyrophosphate (TPP) → Hydroxyethyl-TPP + CO2 (via DXS).

B) Condensation: Hydroxyethyl-TPP + G3P → DXP

NOTE:

• Similar TPP-mediated 2C transfer in Calvin Cycle.

• 2 classes of plant DXSs: class 1 found in green (photosynthetic) tissues.

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2. 
   

A) Isomerization: DXP → 2-C-Methyl-Erythrose-4-Phosphate (via DXP Reductoisomerase (DXR)).

B) NAPDH-Dependant Reduction: 2-C-Methyl-Erythrose-4-Phosphate + NADPH + Mn+ (cofactor) → 2-C-Methyl-D-Erythrose-4-Phosphate (MEP) + NADP+ (via DXR).

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3. 
   

A) CTP-Dependant Cytidyl Trranfer: MEP + Cytidine Triphosphate (CTP) → 4-Diphosphocytidyl-2-C-methyl-D-erythriol (CDP-ME) + PPi (via MEP Cytidyltransferase (MEPC/IspD)).

B) ATP-Dependant Phosphorylation: CDP-ME + ATP → CDP-ME2P + ADP (via CDP-ME Kinase (CMK/IspE)).

C) Cyclization: CDP-ME2P → 2-C-erythriol-2,4-cyclodiphosphate (cMEPP) + CMP (via cMEPP Synthase (MCS/IspF)).

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4. Fd-Dependant Reduction and Linearization: 2 x Ferredoxin (Fd) + cMEPP → 1-hydroxy-1-methyl-2-(E)-butenyl-4-phosphate (HMBPP) + 2 x Fd + H2O (via cMEPP Reductase/HMBPP Synthase (HDS/IspG)).

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5. NADPH-Dependant Reduction: HMBPP + NADPH → IPP + NADP+ + H2O (via HMBPP Reductase (HDR)).

• Favours IPP formation (6:1 IPP to DMAPP), as more products require it (IPI used to convert more if needed).

Isoprenoids - Synthesis (Evidence of Plant MVA + MEP Pathways)

Following transformation by glycolytic enzymes and pyruvate dehydrogenase, isotopically-labelled glucose at C1 appears in methyl groups of:

MEP: labelled pyruvate and G3P (C3) → IPP labelled at C1 and C5.

MVA: labelled acetyl-COA → IPP labelled at C2, C4, and C5.

Isoprenoids - Classes and Examples

1. Prenyl diphosphates.

   • Short-Chain Prenyl Diphosphates - C10 prenyl diphosphates.

   • Medium-Chain Prenyl Diphosphates - C15-25 prenul diphosphates.

   • Long-Chain Prenyl Diphosphates - C30+ prenyl diphosphates.

   
   

2. Terpenes

   • Hemiterpenes - Most basic isoprenoid unit (EX: Isoprene)

   • Monoterpenes - C10 isoprenoids.

     • 3 classes:

       • Acyclic

         • EX:

           • Linalool

           • Citrol

           • Mycene

           • Geraniol

       • Monocyclic

         • EX:

           • Limonene

           • P-cymene

           • Methol

           • Thymol

       • Bicyclic.

         • EX:

           • Eucalyptol

           • Carene

           • Thujeine

           • Pinene

   • Sesquiterpenes - C10 isoprenoids.

     • 3 classes:

       • Monocyclic (EX: Zingiberene)

       • Bicyclic (EX: Casbiene)

       • Tricyclic (EX: Patachoutol)

   • Diterpenes - C20 isoprenoids (EX: Steviol).

   • Long-Chain Isoprenoids - C30+ isoprenoids.

     • Triterpenes - C30 isoprenoids (EX: Digitoxigenin).

     • Polyisoprenoids - C30+ isoprenoids (EX: Dolichol)

Isprenoids - Prenyl Diphosphates (Prenyltransferases)

Prenyltransferases - Enzymes forming prenyl diphosphates by “head-to-tail” condensation of IPP onto DMAPP, beginning with forming and extending a C10 allylic product.

• Classes:

  • Trans-Prenyltransferases (TPTs) - Add IPP in the trans configuration.

  • Cis-Prenyltransferases (CPTs) - Add IPP in the cis configuration.

• Final chain length determined by prenyltransferase specificity.

  • Enzyme named after product formed (EX: Geranyl Pyrophosphate Synthase (GPS) forms geranyl pyrophosphate (GPP)).

Isoprenoids - Prenyl Diphosphates (Synthesis - C10 Prenyl Diphosphates)

A) IPP + DMAPP → Geranyl Diphosphate (GPP) + PPi (via GPP Synthase (GPS)).

• Enzyme location (GPS): Plastid.

• Enzyme type (GPS): TPT.

• Product use (GPP): Monoterpenes.

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B) IPP + DMAPP → Neryl Diphosphate (NPP) + PPi (via NPP Synthetase (NPS)).

• Enzyme location (NPS): Plastid.

• Enzyme type (NPS): CPT.

• Product use (NPP): Monoterpenes.