Chemistry of Biosynthesis

Reading and Literature References

John Mann: Chemical Aspects of Biochemistry, Oxford Chemistry Primer.<
Jonathan Clayden, Nick Greeves, Stuart Warren, Peter Wothers: Organic Chemistry, chapter 50.

Terminology

Primary Metabolism is made up of the fundamental molecules and reactions which are common to all known life forms. Photosynthesis in plants converts carbondioxide into carbohydrates which are converted into energy as adenosin triphosphate (ATP) via the citric acid (Krebs cycle). ATP also drives the biosynthesis of amino acids which are the building blocks of proteins. The building plan for the synthesis of proteins is stored in nucleic acids (DNA and RNA) as genetic information.

Secondary Metabolism is made up of molecules and reactions which are not common to all life forms, but occur in distinct species only. Often secondary metabolites confer a particular advantage to some species. Thus, some fungi produce penicillin as a defense mechanism against other evolutionary competing species. Ironically perhaps, secondary metabolism has been of most interest to organic chemists: The structures of secondary metabolites are extraordinarily complex and their biosynthesis is only beginning to be understood.

Proteins and Enzymes are polypeptides which fulfil both structural and functional roles in cells. Structurally they contribute to the architecture of the cell. Functionally they are involved in the catalysis of reactions. In that role they are also referred to as enzymes. Chemically speaking enzymes and proteins are polypeptides. Inter- and intramolecular forces allow enzymes to adopt distinct three-dimensional shapes. Enzymes can act as catalyst by helping to orientate reactants in a way that facilitates the reaction. Functional groups of individual amino acids provide acidic or basic residues and catalyse the reaction. Sometimes enzymes carry additional groups (Co-factors) to catalyse reactions.

Co-factors (or prosthetic groups) are small non-proteinaceous add-ons to enzymes which extend the capability of enzymes to catalyse reactions. Enzymes containing co-factors are referred to as dependant on them, i.e. PLP-dependant enzymes.

Bio-Molecule Drop Down Menu

The Tool Kit: Organic Chemistry in Biological Systems

Despite the incredible variety and complexity of compounds produced by biological systems, only relatively few reaction types are used to create them. The following will give an overview of the most common reaction types. Since the underlying principles of biological chemistry are the same as those of organic chemistry you will be referred back to relevant sections of other tutorial topics.

Remember: The chemistry of living systems is no different from the chemistry you have encountered so far - just more complex.

This section will cover some important reaction classes which occur in biological systems.To underline that there is no fundamental difference between biological chemistry and organic chemistry the organic counter part of a biological reaction will be given.

Carbon-Carbon Bond Formation

Alkylation Reaction

Aldol and Claisen Reactions

Oxidative Coupling of Phenols

Umpolung

Transamination

Decarboxylation

Reduction

Oxidation

Carbon-Carbon Bond Formation

Alkylation Reactions

Methylation is an important transformation in the biosynthesis of many secondary metabolites. Organic chemists use methyl iodide or methyl sulphonates for methylations. Nature's equivalent is S-adenosyl methionine (SAM). The driving force for methyl group transfer is the conversion of a sulphonium ion into a neutral sulphide.

SAM is biosynthetically derived from the amino acid methionine and ATP. The nucleophilic substitution shown below occurs because triphosphate is an even better leaving group.

SAM is the co-factor of methyltransferase enzymes.

Aldol and Claisen Reactions

Reactions between enolates (and their equivalents) with aldehydes or ketones are referred to as aldol reactions (Tutorial links: Carbonyl Chemistry I, Carbonyl Chemistry II) whereas reaction of enolates with esters are referred to as Claisen reactions. They are the most common method to form carbon-carbon bonds. Nature's equivalents of enolates are enamines and coenzyme A. These are co-factors of aldolase enzymes.

Enamines

The side chain of the amino acid lysine carries an amino group. Reaction with carbonyl compounds leads to imines which tautomerise to give enamines. Enamines are enolate equivalents and react with carbonyl compounds through nucleophilic attack via their b-carbon. They are used in a very similar way in organic chemistry (Tuturial link: Organonitrogen Chemistry) as shown below for the reaction of a secondary amine (pyrrolidin) with a ketone.

Aldol reactions require several levels of control:

• Enol versus carbonyl component: carbonyl compounds with acidic a-protons can either be deprotonated and react as nucleophiles, or react as electrophiles through their carbonyl group. If this is not carefully controlled an intractable mixture of products ("cross-aldol products") is obtained. Formation of an enamine avoids this problem. The enamine is only nucleophilic.
• Regioselectivity: enamines are ambident nucleophiles. They can in principle react through the carbon or the nitrogen atom. For aldol-type processes, only reactions through the carbon atom lead to the desired product. In biological systems the regioselectivity is controled by the steric environment of the enzyme active site.
• Stereoselectivity: The stereochemistry of aldol reactions is highly complex - syn, anti, matched case, mismatched case - are just a few keywords highlighting how difficult it is to control the relative and absolute stereochemistry of aldol products. In biological systems this is again taken care of by the stereochemistry of the active site which serves as a blue-print for the reaction products.

Coenzyme A

Nature's ester enolate equivalent is Coenzyme A. It carries a free thiol group to which carboxylic acid residues are transferred. Thioesters are very reactive intermediates. They are both activated towards nucleophilic attack (electrophilicity of the carbonyl group) and abstraction of a proton (acidity of the a-proton).

The Claisen reaction between acetyl-CoA and malonyl-CoA illustrates how b-keto esters are built up by Nature using the enolate derived from acetyl-CoA as nucleophile.

This reaction plays an important role in the biosynthesis of fatty acids.

Oxidative Coupling of Phenols

Phenols are susecptible to oxidation to quinones. The oxidant can be air, but the presence of Fe(III) catalyses the reaction. The resulting phenoxy radical is mesomerically stabilised by delocalisation into the ortho- and para-position of the aromatic system.

Recombination is a common reaction path to terminate radical reactions (Tutorial link: Reactive Intermediates). Recombination of phenoxy radicals leads also to re-aromatisation through keto-enol tautomerism.

The same recombination can occur in ortho-para and para-para mode.

Umpolung

This term describes reversal of normal polarity at a carbon atom (Tutorial Link: Phosphorous and Sulphur Chemistry). This seemingly exotic trick is used by a number of very important enzymes. The common feature of these enzymes is a thiamin diphosphate co-factor. Chemically speaking, this co-factor contains a thiazolium ring (Tutorial Link: Heteroaromatic Chemistry). The thiazole ring can easily be deprotonated and forms a Zwitter-ionic salt which reacts as a nucleophile through the carbon atom.

Reaction with the a-keto acid pyruvate generates a heterocyclic enol and carbon dioxide. Although the enol is relatively stable it retains its activity because it has lost fully aromatic character.

The enol reacts as a nucleophile with carbonyl compounds. Transfer of the acetyl group restores the aromatic thiazolium system. Note that the acetyl group is formally transferred as an anion with its negative charge on the carbon of the carbonyl group. This is reversed polarity and constitutes an Umpolung.

Transamination

The term transamination refers to the interconversion of carbonyl and amino groups. Condensation of an amine with an aldehyde as shown below gives an imine. What is formally only a tautomerisation reaction converts imine A into its tautomer B which upon hydrolysis yields the "transaminated" products. i.e. the product in which the amine and the carbonyl group have been swapped..

This is a highly simplified view of the transamination reaction.

Firstly, aldehydes do not occur in biological systems due to their chemical instability. The biological equivalent of aldehydes are imines.

Secondly, imines are chemically stable towards this type of tautomerisation reaction. An enzyme is required to effect this transformation and the enzymes employs a co-factor (or prosthetic group).This cofactor is pyridoxal phosphate (PLP).

PLP is attached to the enzyme forms an imine with a lysine residue. This link attaches the co-factor to the enzyme and converts the aldehyde into its biological equivalent, the imine.

The conversion of amino acids into a-keto acids (also sometimes referred to as a-oxo-acids) is a central reaction of primary and secondary metabolism.

In the first step of transamination reactions, pyridoxalphosphate in its biological form of imine is tranferred to the substrate amino acid.

Then the PLP-dependent enzymes catalyses the tautomerisation of the imime.

In the final step, hydrolysis of the imine gives the products.

Note, that pyridoxal phosphate (PLP) has been converted into pyridoxamine by the transamination reaction. A second transamination step is required to convert pyridoxamine back into PLP. This restores the co-factor and the enzyme can carry out another transamination reaction.

Mechanism of PLP-catalysed transaminations

The a-hydrogen of the imine is in conjugation with the protonated pyridinium nitrogen. The positively chareged nitrogen increases the aciditiy of the a-hydrogen and facilitates proton abstraction. The product is an extended conjugated system incorporating both an imine and an enamine.

In the final step, protonation occurs at the d-carbon to the pyridine nitrogen, thus restoring the aromatic system. Hydrolysis of the imine gives the final products.

Chemically speaking, this step could be considered as an acid catalysed vinylogous imine-enamine tautomerisation (Tutorial Link: Organonitrogen Chemistry).

Decarboxylation

Decarboxylation reactions are important in biological systems because intermediates which are chemically disposed for decarboxylation, such as b-keto acids, occur frequently in primary and secondary metabolism.

a-Keto acids are chemically not predisposed towards decarboxylation. This is reflected in much higher temperatures required to effect the above transformation. Nature uses enzymes for this reaction which carry PLP as co-factor. The schemes below shows the decarboxylation of an a-amino acid.

The amino acid is bound to to PLP as the imine in the first step.

In the actual decarboxylation step, the electronic effects are the same: the pyridine nitrogen acts as an electron-withdrawing group, this time facilitating deprotonation of the carboxylic acid group. Loss of carbondioxide and hydrolysis of the imine gives the reaction products.

Reduction

The biological equivalent of hydride transfer reagents, such as NaBH4, is nicotinamide adenine dinucleotide (NADH) and its phosphorylated analogue NADPH. These are co-factors of reductase enzymes. The stick model of NAD is taken from an actual x-ray crystallographic analysis of human alcohol dehydrogenase enzyme (PDB entry: 1HT0)

The pyridinium ring acts as hydride acceptor in the oxidation step, whilst 1,4-dihydropyridine system acts as hydride donor in the reduction step.

The stereoselectivity of the reduction step relies on the "chiral environment" provided by the active side of the enzyme. NADH is a co-factor which is held in the acitve site of the enzyme (alcohol dehydrogenase in this case) by non-covalent interactions. The image below shows NADH and amino acids in a distance of 5 Å from NADH.

The image on the left is a close-up view of the residues neighbouring NADH in the active site. The image on the right shows the whole enzyme (the enzyme is actually a dimer and only one half is shown for clarity). The residues making up the active site are shown in CPK mode.

Oxidation

NAD-dependant Enzymes

Oxidation is the reverse of reduction and the oxidised form of NADH can act as an oxidant. In oxidation-mode NAD/NADH-dependant enzymes are referred to as oxidase enzymes. This form is called NAD. In fact, NAD and NADH have to be reversible redox pairs to allow the co-factor and the enzyme to act as true catalysts.

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Cytochrome-P450-dependant Enzymes

The redox-active species in this class of enzymes is the Fe(III)-Fe(II) couple. The iron centre is coordinated to a porphorine system. Together they form the haem co-factor of oxygenase enzymes (note the difference to oxidase enzymes which contain NAD as co-factor). The name cytochrome P450 is due to the strong absorption at 450nm of enzymes that contain a haem co-factor when co-ordinated to carbon monoxide.

Non-Haem a-Keto-glutarate-dependant Oxygenases

Enzymes belonging to this class contain an iron centre, but no haem co-factor. Isopenicillin-N-synthase, the crucial enzyme in the biosynthesis of penicillin belongs to this class.