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SECONDARY METABOLISM: The Building Blocks and Construction Mechanism Primary and Secondary Metabolism

intermediary metabolism-an integrated network of enzyme-mediated and carefully regulated chemical reaction metabolic pathways- pathways involved in intermediary metabolism primary metabolism (biochemistry)- processes that demonstrate the fundamental unity of living matter wherein crucially important molecules such as carbohydrates, proteins, fats and nucleic acids are modified and synthesized -processes involves the synthesis, degradation and interconversion of compounds commonly encountered in all organism Carbohydrates and sugar degradation Krebs/citric acid/TCA cycle- release of energy from organic compounds by oxidative reactions Fatty acid oxidation -oxidation- provides energy oxidative phosphorylation- further process for aerobic organisms to optimize fatty acid oxidation primary metabolites- compounds involved in primary metabolism secondary metabolism (natural products chemistry)- metabolism concerned with compounds which have a much more limited distribution in nature secondary metabolites- compounds that are found in only specific organisms or groups of organisms and are an expression of the individuality of species -not necessarily produced under all conditions -provides most of the pharmaceutically active natural products -in vast majority of cases, the function of these compounds and their benefit to the organism are not yet known -are produced for reasons e.g., *as toxic materials providing defense against predators *as volatile attractants towards the same or other species, or *as coloring agents to attract or warn other species

The Building Blocks

BUILDING BLOCKS in biosynthesis of secondary metabolites: Acetyl CoA - formed by oxidative decarboxylation of the glycolytic pathway product pyruvic acid -also produced by the -oxidation of fatty acids -Acetate pathway produces the secondary metabolites: phenols prostaglandins macrolide antibiotics various fatty acids and derivates at the primary-secondary metabolism interface Shikimic acid -produced from a combination of phosphoenolpyruvate, a glycolytic pathway intermediate, and erythrose 4-phosphate from pentose phosphate pathway -pentose phosphate pathway: may be employed in reactions that degrades glucose but also feature in the synthesis of sugar by photosynthesis -shikimic pathway leads to a variety of: phenols cinnamic acid derivatives lignans alkaloids

Mevalonic acid -formed from three molecules of acetyl-CoA -mevalonate pathway channels acetate into a different series of compounds than does the acetate pathway Methylerythritol phosphate -arise from a combination of two glycolytic pathway intermediates, pyruvic acid and glyceraldehyde 3-phosphate by way of deoxyxylulose phosphate -together with mevalonate, are responsible for the biosynthesis of a vast array of: terpenoid steroid Other building blocks based on amino acids -peptides, proteins, and alkaloids of many antibiotics are derived from amino acid -Glycolytic pathway and Krebs cycle- used in constructing many amino acids; ornithine and lysineimportant alkaloid precursor -Shikimate pathway- produces aromatic amino acids such as phenylalanine, tyrosine, and tryptophan

C1- the simplest building block; in the form of methyl group -most frequently attached to oxygen or nitrogen, but occasionally to carbon or sulfur -derived from the S-methyl of L-methionine -ex: methylenedioxy group (-OCH2O-)

C6C2 or C6C1- represents shortened forms of C6C3 system

C2- two carbon unit may be supplied by acetyl-CoA -may be a simple acetyl group (ex. ester) or part of a long alkyl chain (ex. fatty acid), or part of an aromatic system (ex. phenols) -ex: acetyl-CoA first converted to more reactive malonyl coA

C6C2N- formed from either L-phenylalanine or L-tyrosine (common precursor) -carboxyl carbon of the amino acid is removed

C5- branched-chain isoprene unit -formed from: -mevalonate- is the product from three acetyl-CoA molecules, but only five of its six carbons are used since the carboxyl group is lost -methylerythritol phosphate- formed from a straight-chain sugar derivative, deoxyxylulose phosphate, which undergoes a skeletal rearrangement to form the branched-chain isoprene unit

Indole.C2N- derived from decarboxylation of L-tryptophan

C4N-found as a heterocyclic pyrrolidine system; produced from the non-protein amino acid L-ornithine which supplies not its -amino nitrogen, but the -amino nitrogen -the carboxylic acid function and the amino nitrogen are both lost

C6C3- phenylpropyl unit obtained from the carbon skeleton of either L-phenylalanine or L-tyrosine, shikimate-derived aromatic amino acids -requires the loss of amino group -C3 side chain may be saturated, unsaturated, or oxygenated; it may sometimesbe cleaved

C5N- produced in exactly the same way as the C4N unit, but using L-lysine as precursor -the -amino nitrogen is retained, and the unit is commonly found as a piperidine ring system

The Construction Mechanism

1. Amino Acids and Transamination -amination of 2-oxoglutaric acid (Kreb cycle intermediate) to glutamic acid: a reductive amination responsible for amino acid synthesis

-Glutamic acid: formation of other amino acids via transamination -the exchange of the amino group from an amino acid to a keto acid -provides the most common process for introduction of nitrogen into amino acids and emoval of nitrogen from them -catalyzed by pyridoxal phosphate (PLP) 1) formation of imine intermediate (aldimine) 2) protonation of pyridine nitrogen: provides an electron sink and makes the -hydrogen more acidic 3) generation of dihydropyridine ring system 4) reprotonation produces a new imine (ketimine) and restoration of aromaticity in the pyridine ring

-Glutamic acid-2-oxoglutaric acid couple provide usual donor-acceptor molecules for the amino group -glutamate transaminase- most important transaminase -amino group is transferred from glutamic acid to 2-oxo-glutaric acid 2. Decarboxylation reactions -degradative modification in losing carbon atoms -feature of biosynthetic utilization of amino acids -amino acids -PLP-dependent reaction -protonated nitrogen acts as electron sink -conjugated system allows loss of carboxyl proton with subsequent breaking and loss of CO2 -after protonation of original -carbon, amine is release from the coenzyme by hydrolysis; PLP regenerated

-keto acids -thermally labile and rapidly decarboxylated in vitro via a cyclic mechanism -enol form ketone -phenolic acid: its carbonyl carbon group in its keto tautomer activates system for decarboxylation -keto acids -its decarboxylation is a feature of primary metabolism -ex: glycolysis= pyruvic acid acetaldehyde entry for Krebs cycle= pyruvic acid acetyl CoA -thiamine diphosphate TPP depended; coenzyme containing thiazole ring -1) proton in thizolium ring is acidic removed carbanion (ylid, species with + and charges on adjacent atoms; acts a nucleophile) 2) carbonyl group of pyruvic acid 3)decarboxylation (N+ as electron sink) 4) formation of enamine 5) Tautomerism to the iminium cation is followed by a reverse aldol reaction, with regeneration of ylid 6) oxidation step: involves enzyme-bound disulfide containing coenzyme lipoic acid 7) formation of intermediate enmaine which attack a sulfur in lipoic acid moiety with S-S bond fission 8) regeneration of TPP ylid by reverse aldol 9) acetyl group released as acetyl-CoA by displacement with thiol coenzyme A 3. Oxidation and Reduction Reactions -classified according to the type of enzyme involved and their mechanism of action Dehydrogenases -remove two hydrogen atoms from the substrate, passing them to a suitable coenzyme acceptor -PYRIDINE NUCLEOTIDE: nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide (NADP+) serves as hydrogen acceptor -one hydrogen from the substrate (that bonded to carbon) is transferred as hydride to the coenzyme and the other (as proton) is passed to the medium

- LiAlH4 or NaBH4 : complex metal hydrides used in reduction reaction -NADH and NADPH : hydride-donating reducing agents

-flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) coenzyme used as acceptor (of two hydrogen atoms) in an oxidative process

-important in primary metabolism in liberating energy from fuel molecules in the form of ATP -the reduced coenzymes formed are reoxidized via ETC of oxidative phosphorylation H2O oxidases -remove hydrogen from a substrate, and pass it to molecular oxygen or to hydrogen peroxide and form water

-ex: peroxidase oxidases that uses H2O2 monooxygenase -catalyzes the addition of one oxygen atom from molecular oxygen to the substrate -the second oxygen atom from O2 is reduced to water by an appropriate hydrogen donor, e.g. NADH, NADPH, ascorbic acid -> mixed function oxidase since they may be considered oxidases in this respect -cytochrome P-450 dependent monooxygenasesinvolved in biological hydroxylations (metabolismof foreign compounds such as drugs/ hydroxylases and mammalian detoxification) -inhibited by CO -contains an iron-porphyrin complex (haem) which is bound to the enzyme -Fe redox during binding and cleavage of oxygen atoms, w/ subsequent transfer of one atom to the substrate -Sample reactions:

NIHShift -aromatic hydroxylation with arene oxide (epoxide) intermediates -epoxide opens up, hydrogen atom originally attached to the position which becomes hydroxylated transfers to the adjacent carbon on the ring -enolization loss of some of the hydrogen atoms

oxidative cyclization -of o-hydroxymethoxy-subtituted aromatic system giving a methyenedioxy group -yield hemiacetal of formaldehyde cyclization methylenedioxy bridge by ionic mechanism

dioxygenase -introduces both atoms from molecular oxygen into the substrate -involed in cleavage of C-C bonds, including aromatic rings -intermediates: cyclic peroxides/dioxetanes -oxidative cleavage of aromatic rings: catechol (1,2-dihydroxy) or quinol (1,4-dihyroxy) substrates cleavage between two adjacent hydroxyls giving products containing aldehyde and/or carboxylic acid functionalities amine oxidase -those which transform an amine into an aldehyde -monoamine oxidases: utilizes flavin nucleotide (typically FAD) and molecular oxygen -involves initial dehydrogenation to an imine -hydrolysis to aldehyde and ammonia -diamine oxidases: require a diamine substrate and oxidize at one amino group using molecular oxygen to give aldehyde -other products: ammonia, H2O2 Baeyer villager monooxygenase -chemical oxidation of ketones by peracids -yields an ester (ketone ester)

-migration of an alkyl group from the ketone -involves NADPH and O2 required by FAD-dependent monooxygenase; leads to formation of a flavinperoxide 4. Phenolic Oxidative Coupling -process readily rationalized by means of radical reactions brought about by oxidase enzymes (peroxidase and laccase systems) known to be radical generators -catalyzed by other enzymes characterized as cytochrome P-450-dependent protein that requires NADPH and O2 cofactors , but no oxygen in incorporated into the substrate

-phenol derived radical formed do not propagate radical chain reaction but undergoes coupling with other radicals dimeric systems -final product is derived by enolization that restores aromaticity to the rings 5. Glycosylation Reaction Aglycone: nonsugar component -processes of attaching sugar units Glycone: sugar component to a suitable atom of an aglycone to give a glycoside, or to another sugar giving polysaccharide -linkages tend to be through oxygen (but not restricted), S-, N-, C -may be a simple nucleophilic displacement reaction of SN2 type -Biosynthesis of glycoside principal pathway: 1. Transfer of a uridylyl group from UTP to a sugar 1-P catalyzed by uridylyl transferases to form UDP-sugar Glucose 1-P + UTP UDP-Glucose + PPi 2. Transfer of the sugar from uridine diphosphate to a suitable acceptor (agylcone) mediated by glycosyl transferases to form glycoside UDP-Glucose + Acceptor (aglycone) Acceptor-sugar (glucoside) + UDP -agent of reaction: nucleoside diphosphosugar (UDP glucose)
GLYCOSIDE: compounds that yield one or more sugars among the products of hydrolysis, with -Dglucose as the most frequently occurring sugar -sugar ethers -glucose=glucoside -acetals in which the hydroxyl of the sugar is condensed with a hydroxyl group of a non-sugar component, and the secondary hydroxyl is condensed is condensed within the sugar molecule itself to form an oxide ring

-uridine: is the nucleoside most frequently employed -configuration (depends on the stereoconfiguratioon of the glycosidic linkage) -configuration: most common found in natural glucosides (resulted when UDP glucose has its leaving group in the -configuration); the only one that occurs in plants -configuration: originated via a double SN2 process (sucrose and starch)

-C-glycosides: a suitable nucleophilic carbon is required (ex. aromatic systems activated by phenol group) -C-glycosylation introduces new carbon-carbon linkage and cleavage requires oxidation, not hydrolysis

- hydrolysis of glycosides is achieved by specific hydrolytic enzymes, e.g. -glucosidase for -glucosides and -galactosidase for -galactosides which mimics the readily achieved acid-catalyzed processes -- and - anomeric hemiacetal forms in an equilibrium mixture results under acidic condition which can interconvert via the open-chain sugar -O, N-,and S-glycosides may be hydrolyzed by acid and C-glycosides are acid stable

Some Vitamins Associated with Construction Mechanism

Vitamin B1~thiamine -water soluble vitamin with a pyrimidinylmethyl-thiazolium structure -available in diet with cereals, beans, nuts, eggs, yeast, vegetables, wheat germ and yeast -dietary deficiency leads to beriberi, characterized by neurological disorders, loss of appetite, fatigue, and muscular weakn ess -as thiamine diphosphate, is a coenzyme for pyruvate

dehydrogenase, which catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA, and for 2oxoglutarate dehydrogenase, which catalyses a similar reaction on 2-oxoglutarate in the Krebs cycle -as cofactor for transketolase, which transfers a two-carbon fragment between carbohydrates in the pentose phosphate pathway; important for carbohydrate metabolism -stable in acid solution but decomposes above pH 5 and normal cooking heat Vitamin B2~riboflavin -water-soluble vitamin with an isoalloxazine ring linked to Dribitol -available in food: liver, kidney, dairy products, eggs, meat and vegetables , yeast -stable in acid solution and does not decompose when cooked but sensitive to light -may be produced synthetically or by fermentation using yeastlike fungi Eremothecium ashbyii and Ashbya gossypii - deficiency (though uncommon) causes skin problems and eye disturbances -components of FMN and FAD, coenzymes which play a major role in oxidation-reduction reactions -contains ribitol, not ribose, in its structure Vitamin B3~ nicotinic acid, niacin -stable, water-soluble vitamin found in meat, liver, fish, wheat germ and yeast -nicotinic acid: may be provided by amino acid tryptophan; is converted into nicotinamide; may lower cholesterol and TAG concentration by inhibiting synthesis -nicotinamide: may be in the form NAD+ and NADP+, needed for oxidation-reduction reaction, and as carrier in primary metabolism; more preferred in supplements than nicotinic acid that has a higher risk of vasodilation -deficiency: pellagra (manifests in diarrhea, dermatitis, dementia), oral lesion and a red tongue Vitamin B5~pantothenic acid -water-soluble vitamins found in yeast, liver, cereals -important in metabolism as a part of the structure of coenzyme A- the carrier molecule essential for carbohydrate, fat, and protein metabolism -implicated in enzymes responsible for the biosynthesis of fatty acids, polyketides, peptides Vitamin B6~pyridoxine (pyridoxol), pyridoxal and pyridoxamine -Pyridoxine: predominating in plant materials -Pyridoxal, pyridoxamineL main forms in animal tissues -Pyridoxal 5-phosphate: coenzyme for a large number of enzymes, particularly those involved in amino acid metabolism (transamination and decarboxylation) -central role in production of neurotransmitters: serotonin, noradrenaline and histamine, gamma aminobuyric acid (from glutamic acid) -sources: meat, salmon, nuts, potatoes, bananas, cereals -deficiency may result due to malabsorption, induce by some drug treatments (antagonist or increase excretion of vitamins)

-symptoms of deficiency: similar to vit. B3 and B2, including eye, mouth, nose lesion and neurological changes Vitamin B12~cobalamins -based on complex structure on a corrin ring, which has two of the pyrrole rings directly bonded, central atom is cobalt -structure: four of the six coordinations are provided by the corrin ring nitrogen atoms, the fifth by a dimethylbenzimidazole moiety, the sixth is variable (cyano in cyanocobalamin /vitamin B12, hydroxyl in hydroxocobalamin/vitamin B12a, or other anions may feature -5-deoxyadenosylcobalamin (coenzyme B12): physiologically active coenzyme form of the vitamin -microbial origin, with intestinal flora contributing towards human dietary needs -food source: liver, kidney, meat, seafood -pernicious anemia: due to insufficient vitamin B12, lack of gastric glycoprotein (intrinsic factor) so facilitate its absorption that results in nervous disturbances and low production of red blood cells -Hydroxocobalamin has longer lifetime than cyanocobalamin -coenzyme B12 : is a cofactor for conversion of methylmalonyl-CoA to succinyl-CoA, oxidation of fatty acids with odd number of C atoms, and for methylation of homocysteine to cysteine

Vitamin H~Biotin -functions as carboxyl carrier, binding CO2 via a carbamate link, then donating this in carboxylase reactions -ex: carboxylation of acetyl-CoA to malonyl-CoA, of propionyl-CoA to methylmalonyl CoA and of pyruvate to oxaloacetate during gluconeogenesis -found in eggs, liver, kidney, yeast, cereals, milk -produced by intestinal microflora -deficiency is rare and can be triggered by a diet rich in raw egg whites which contains avidin(binds tightly with biotin) -deficiency symptoms: dermatitis and hair loss