Terpenes and Fatty Acids

• Phosphates and pyrophosphates are common biological leaving groups. Acyl groups are activated for acylation reactions by forming phosphoric anhydrides (making acyl phosphates). This occurs in aqueous medium (cellular conditions) and can give hydrolysis as an unwanted side reaction.

  
• Cytochrome P450:

  • Family of enzymes
  • Use molecular oxygen to oxidise substrates e.g., light alkanes
  • Formation of peroxides, thiols and amines
• Gibberellin biosynthesis constitutes multiple oxidations at a single site with kaurene oxidase (P450) using the cofactor NAD+.
• Haemoglobin contains Fe (III) that is reduced to Fe (II) which binds to molecular oxygen.

  • Forms a covalent bond, then a radical, then peroxy-intermediate
  • Fe (V) ion formed is a ferryl-intermediate, a powerful oxidising agent
  • The radical rebound step removes the oxygen, returning haem to its resting state

  
• Basic terpene synthesis:

  1. Claisen-like reaction
  2. Aldol-like reaction
  3. Decarboxylation

  
• Monoterpene biosynthesis:

  • Carbocation formation that leads to initiation, propagation and termination reactions
  • C5 unit reactions that include isomerisation from IPP (isopentyl pyrophosphate) to DMAPP (dimethylallyl pyrophosphate)
  • Phosphorylation requires ATP and is catalysed by kinases

  
• Classes of terpenes:

  • C10 = monoterpene
  • C15 = sesquiterpenes
  • C20 = diterpenes
  • C30 = triterpenes
• The formation of terpenes from the C5 monomer is done by two reactions:

  1. Carbocation formation
  2. C-C bond formation

  
• The extension of C5 units lead to:

  • Geranyl = C10
  • Farnesyl = C15
  • Geranylgeranyl = C20

  
• C30 terpenes are formed by the joining of two C15 units.

  • Produces squalene
  • Squalene is stereoselective

  
• The alpha-terpinyl cation is a common intermediate that can be converted to different monophosphates using enzymes e.g., squalene synthase.

  • Reasonably stable precursor
  • Linalyl phosphate intermediate has single bond character, allowing for rotation and formation of stereoisomers
  • Geranyl phosphate adopts the chair conformation

  
• Deprotonation to a cyclopropane unit is catalysed by 3-carene synthase, followed by a rearrangement via hydride shift.

  
• Trichodiene (C15):

  • Biosynthesised by Fusarium sporotichioides
  • Synthesis begins with activation by formation of tertiary nerolidyl pyrophosphate
  • The pyrophosphate group is situated between Mg2+ cofactors

  
• Gibberellins:

  • Plant hormones controlling growth and development
  • Some pathogens produce gibberellins to weaken the host
  • Mutant strains with modified gibberellin metabolism result in dwarf and giant strains
• Biosynthesis of gibberellins:

  1. Electrophilic cyclisation from geranylgeranyl phosphate to copalyl pyrophosphate
  2. Formation of kaurene from copalyl phosphate from a second cyclisation
  3. Biosynthesis with several oxidase/kinase enzymes

  
• Geranylgeranyl pyrophosphate adopts the chair-chair conformation.

  
• Cofactors are required for fatty acid biosynthesis:

  • Living organisms use Coenzyme A thioesters
  • CoA is a thiol and forms thioesters
  • Thioesters are more reactive than normal (oxy-) esters but more stable to hydrolysis than acyl phosphates

  
• Synthesis of CoA thioesters:

  1. Activation of the acid as the acyl adenylate
  2. Reaction with CoA to make the thioester

  
• Acyl adenylate is an intermediate that is unstable to hydrolysis and remains bound to the active site throughout. The use of thioesters is to control the reactivity of carbonyl compounds.

  • Controlled by the δ+ on the carbon
  • More efficient delocalisation of the lone pair onto the double bond results in less reactivity
  • Resonance structures are related to keto-enol tautomerism

  
• Acyl carrier proteins (ACP)

  • Small proteins/domain attached to a larger protein
  • Contains a flexible pantetheine arm derived from CoA
  • Arm attached to serine residue in the peptide backbone

  
• Fatty acid synthesis:

  1. Thioester formation
  2. New C-C bond formation
  3. Ketone reduction
  4. Dehydration
  5. Enoyl reduction
  6. Thioesterase

  
• The reaction of fatty acid synthesis are catalysed by fatty acid synthase (FAS) which has several active sites, each of which catalyses a different step. In higher organisms, each of the active sites are found in their own special region of the FAS known as a domain.

  
• Step 1 of fatty acid synthesis:

  • Acyl CoA activation
  • Acetyl group is loaded onto the active site on the ketosynthase (KS) domain
  • A malonyl group is loaded onto an acyl carrier domain

  
• Step 2 of fatty acid synthesis:

  • Malonyl group is activated by malonyl acyl transferase (MAT)

  
• Step 3 of fatty acid synthesis:

  • C-C bond formation catalysed by beta-ketoacyl synthase (KS)
  • The ACP:KS:AT complex has KS as a dimer, KSa and KSb

  
• Step 4 of fatty acid synthesis:

  • Reduction of the ketone by NADH
  • Catalysed by ketoreductase (KR)

  
• Step 5 of fatty acid synthesis:

  • Dehydration of the alcohol to remove water
  • Catalysed by dehydratase (DH)
  • Via a E1cB mechanism

  
• Step 6 of fatty acid synthase:

  • Reduction of the alpha-beta unsaturated thioester
  • Catalysed by enoyl reductase (ER) with cofactor NADH

  
• Step 7 of fatty acid synthesis:

  • Release of the fatty acid chain
  • Catalysed by thioesterase
  • If the chain is not long enough it is transferred back to the active site of KS to allow the cycle to repeat

  
• Structure of fatty acid synthase:

  • Identified by X-ray crystallography
  • Dimeric with 2 fold symmetry
  • ACP is mobile

  
• The cofactor biotin is used for the formation of malonyl-CoA.

  
• Polyketide synthases:

  Type I PKS: large, highly modular proteins present in eukaryotes

  Type II PKS: aggregates of monofunctional proteins present in prokaryotes

  Type III PKS: small and do not use ACP domains - rely on CoASH and Cys
• Polyketide synthesis involves the assembly of the back bone using KS. The first cycle makes a beta-keto group, the second cycle gives an addition to this group. Depending on the folding of the chain, functional groups can be generated.

  
• Orsellinic acid is formed by an aldol condensation.

  • 6 membered ring is thermodynamically favoured
  • Final step is the hydrolysis of the thioester
  • Acidic protons alpha to 2 carbonyls are the most reactive to base

  
• Alternative folding of the precursor to orsellinic acid gives phloracetophenone.

  
• Carbonyls can be partially reduced to a double bond to allow for ring formation.

  • A second ketosynthase step as followed by KR and DH that introduce the enoyl group

  
• Neocarzinostatin:

  • Enediyne antibiotic produced by streptomyces carzinostaticus
  • Kills microbes by damaging DNA
  • Epoxide ring opening gives cyclisation followed by homolytic fission to produce radicals

  
• Complex aromatic polyketides:

  • Tetracyclines are used in chemotherapy e.g., daunomycin
  • Requires 8 steps for oligomerisation
  • Needs post-synthetic modification

  
• Type III polyketide synthases are small with iterative KS domain.

  
• Erythromycin A:

  • Antibiotic, aliphatic polyketide
  • Starter unit is propionyl CoA
  • Methylmalonyl CoA used for methyl substituents
  • Cyclic product is formed by condensation to give an ester