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Plant BiochemistryLecture 11: AMINO ACIDS II

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LEARNING OUTCOMESStudents, after mastering materials of the presentlecture, should be able

1. to explain the biosynthesis of aromatic aminoacids including pathways, enzymes and the siteof synthesis.

2. to explain the biosynthesis of glutamate-derivedamino acids.

3. to explain the biosynthesis of

4. to explain the biosynthesis of histidine aminoacid

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LECTURE OUTLINE

3. AROMATIC AA1. Synthesis of chorismate2. Chorismate mutase3. Phenylalanine and Tyrosine4. Tryptophan Biosynthesis5. Anthranilate Synthase6. PAT, PAI and IGPS Enzyme7. Tryptophan synthase8. Biosynthesis in Plastids9. Stress Conditions

4. ASPARTATE-DERIVEDAA

1. Biosynthetic Pathways2. Sulfur Requirement3. Biosynthetic Regulation

4. Lysine Biosynthesis andDegradation

5. BRANCH-CHAIN AA1. Threonine Deaminase2. AHAS and IPMS

6. GLUTAMATE-DERIVEDAA1. Proline Metabolism2. Metabolism Regulation3. Arginine Biosynthesis

7. HISTIDINE

3. AROMATIC AA All living cells require the aromatic amino acids

(phenylalanine, tryptophan, and tyrosine) for proteinbiosynthesis.

In plants, the aromatic amino acid pathways alsoprovide precursors for the production of numerousaromatic primary and secondary metabolites such asplant hormones (auxin, and salicylic acid), pigments(anthocyanins), volatiles, defensive phytoalexins, feedingdeterrents (tannins), UV protectants (flavonoids), signalmolecules (isoflavonoids), bioactive alkaloids, andstructural components (lignin, suberin, and cellwall‐associated phenolics) (Fig 7.16).

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Fig 7.16 Synthesis of the aromatic amino acids in plants.In addition to their functions in proteins, phenylalanine, tyrosine,and tryptophan serve as precursors for the synthesis of numerousprimary and secondary metabolites.

The importance of these pathways in plants isunderscored by the fact that about 30% of the fixedcarbon can flow through the common aromatic aminoacid pathway, largely to make lignin.

Owing to their pharmacological and biological activities,aromatic amino acid‐derived plant natural products arewidely used in medicine (e.g., condensed tannins andmorphine) and as food supplements (e.g., flavonoids andtocopherols).

The aromatic amino acid pathways are absent inanimals; the enzymes of these pathways, therefore,became targets for the development of antibioticsagainst human and animal pathogens and plantherbicides.

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1. Synthesis of chorismate Synthesis of chorismate is the common aromatic

amino acid pathway.- Chorismic acid, the final product of the shikimate pathway, is

the last common precursor in the synthesis of the threearomatic amino acids (phenylalanine, tryptophan, andtyrosine) as well as p‐aminobenzoic acid (a precursor for theC carrier tetrahydrofolate), the electron transport cofactorphylloquinone, other naphtho‐ and anthraquinones, andsalicylic acid.

Plastids have a prominent function in this biosynthe-sis, because a full set of the shikimate pathwayenzymes has been localized to these organelles.

The seven‐step synthesis of chorismate via theshikimate pathway (Fig 7.17) begins with thecondensation of two intermediates of carbohydratemetabolism.

The abbreviations SHKA through SKKH are notations proposed forthe genes encoding shikimate pathway enzymes in plants.

Fig 7.17 Chorismatebiosynthesis fromphosphoenolpyruvate anderythrose 4-phosphate.

The two interme-diates are phos-pho enolpyru-vate (PEP) fromglycolysis anderythrose 4-phosphate fromthe pentose phos-phate pathway.

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This reaction is catalyzed by 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase.

Based on the absence or presence of certain domainsin the protein as well as amino acid sequencesimilarity, DAHP synthases have been classified intotwo types (type I and II) with less than 10% sequenceidentity.- E. coli DAHP synthase belongs to the type I DAHP synthase

family with molecular masses less than 40 kDa. Type IIenzymes (≈50 kDa) were first identified in plants, but were lateralso identified in some microorganisms.

The penultimate enzyme of chorismate biosynthesis,5‐enolpyruvylshikimate‐3‐phosphate (EPSP)synthase, catalyzes the reversible production ofEPSP and phosphate from shikimate 3‐phosphateand PEP.

EPSP synthase is the best studied enzyme of theshikimate pathway, as it is the prime target of thecommercially important herbicide glyphosate (Fig7.18).

The final enzyme of thepathway, chorismatesynthase, catalyzesthe elimination of phos-phate and a hydrogenatom from EPSP to pro-duce chorismate(Fig 7.17)

Fig 7.18 Glyphosate, herbicidal consti-tuent of the commercial herbicideRoundup® is a competitive inhibitor ofEPSP synthase

Genes encoding chorismate synthases have been iso-lated from a number of plant species. The tomato(Solanum lycopersicum) genome contains two choris-mate synthase genes that are differentially expressed.

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2. Chorismate mutase Chorismate mutase is the committing enzyme in

phenylalanine and tyrosine synthesis. The biosynthesis of phenylalanine and tyrosine may

occur via two alternative pathways with eitherarogenate or phenylpyruvate/p-hydroxyphenylpyru-vate as intermediates (Fig 7.19).

Recent genetic evidence suggests that the arogenateroute is predominant in plants. This means that plantsdiffer from enteric bacteria and fungi, which primarilyuse the phenylpyruvate route.

Chorismate mutase (CM), the committing enzyme forphenylalanine and tyrosine biosynthesis, catalyzes theintramolecular rearrangement of the enolpyruvyl sidechain of chorismate to produce prephenate.

Fig 7.19 Phenylalanine and tyrosine are predominantly synthesized fromarogenate in plants. This is in contrast to the major pathway inSaccharomyces cerevisiae and E. coli, where phenylpyruvate and4‐hydroxyphenylpyruvate are intermediates.

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In many plants, this activity exists in two isoenzymeforms, CM1 and CM2, which are regulated quitedifferently from one another.

The plastid localization and regulatory behavior ofCM1 are consistent with a role for this activity as acommitting enzyme in an amino acid biosyntheticpathway.

CM1 is feedback‐inhibited by each of the endproducts, phenylalanine and tyrosine, and is activatedby tryptophan, the product of the other branch of thepathway. Tryptophan reverses the inhibition byphenylalanine or tyrosine (Fig 7.20).

This mechanism regulates flux into the two competingpathways by increasing synthesis of phenylalanineand tyrosine when tryptophan is plentiful.

FIGURE 7.20 Allosteric regulation of chorismate mutase controls fluxfrom chorismate into phenylalanine and tyrosine.

When the supply of these amino acids is adequate,the synthesis of phenylalanine and tyrosine is andsuppressed.

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3. Phenylalanine and Tyrosine In plants, the pathway that synthesizes phenylalanine

and tyrosine is regulated by its final reactions. The biosynthesis of phenylalanine and tyrosine in

plants via the arogenate pathway represents anunusual case, wherein the committing reactions arealso the last steps of the pathways.

The final enzyme of phenylalanine biosynthesis isarogenate dehydratase, which catalyzes thedecarboxylation and dehydration of arogenate.- Multiple genes encoding arogenate dehydratase have been

isolated from a number of plant species.- These plant genes encode monofunctional dehydratases that

contain two domains: a catalytic domain and a C‐terminalregulatory domain that is involved in the allosteric regulationby phenylalanine.

Arogenate dehydrogenase (ADH) catalyzes theoxidative decarboxylation of arogenate to tyrosine.

This NADP-dependent enzyme lacks an additionaltyrosine‐binding domain, independent of the substratebinding site (unlike arogenate dehydratase), but it isfeedback‐inhibited by tyrosine competitively withrespect to the arogenate substrate.

ADH activities have been detected in a variety of plantspecies, however, genes encoding these enzymeshave been isolated only from Arabidopsis and maize(two and four, respectively).

Biosynthesis of phenylalanine and tyrosine can alsooccur via the alternative phenylpyruvate andp‐hydroxyphenylpyruvate routes, respectively, inwhich aromatization precedes the transaminationreaction (see Fig 7.19), which then is the final step.

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4. Tryptophan Biosynthesis Conversion of chorismate to tryptophan has

significance beyond amino acid biosynthesis. Plants use this pathway to produce precursors for

numerous secondary metabolites, including thehormone auxin, indole alkaloids, phytoalexins, cyclichydroxamic acids, indole glucosinolates, andacridone alkaloids (Fig 7.22).- These metabolites serve as growth regulators, defense

agents, and signals for insect pollinators and herbivores.Some of these alkaloids, including the anticancer drugsvinblastine and vincristine, have great pharmacological value.

The biosynthetic pathway for tryptophan was the firstproven to be amenable to detailed molecular geneticanalysis (Box 7.2) that results in a generally acceptedin vivo pathway (Fig 7.23).

Genes for all of the enzymes have beencharacterized, and mutants have been identified for

Genes for all of the enzymes have beencharacterized, and mutants have been identified forall but one of the seven proteins (indole‐3‐glycerol‐phosphate synthase; IGPS; an antisenseapproach was chosen to downregulate this enzymein Arabidopsis).

Fig 7.22 The indolering (highlighted inyellow) derives fromthe amino acidtryptophan and is acommon feature ofmany secondarymetabolites in plants

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Fig 7.23 Tryptophan biosynthetic pathway of Arabidopsis. The wild‐type genenames (e.g., ASA1) and mutant designations (e.g., trp5) are indicated to theleft of the arrows.

5. Anthranilate Synthase Anthranilate synthase (AnS) is an amino‐accepting

chorismate‐ pyruvate lyase that catalyzes the firststep in tryptophan biosynthesis, the formation ofanthranilate. It is a two‐subunit enzyme in plants thatfunctions as an α2β2 complex.

The α subunit catalyzes the amination of chorismateand removal of the enolpyruvyl side chain (yieldingpyruvate), acting in concert with the glutamineamidotransferase activity of the β subunit (Fig 7.24).

Analysis of the crystal structure of the bacterialenzyme suggests that the binding of chorismate tothe α subunit triggers a conformational change to anactive state and creates an intermolecular tunnel forammonia transfer from the β to α subunit.

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Fig 7.24 Anthranilate synthase catalyzes the first step of tryptophan biosyn-thesis. Note that the γ‐amide group of glutamine becomes the aromaticamino group of anthranilate. This activity, often called a glutamine amino-transferase, is therefore correctly called a glutamine amidotransferase. Glu-tamine amidotransferases, which are PLP‐independent, generally catalyze areaction in which ammonia generated by hydrolysis of glutamine is chan-neled to a second active site where it acts as a nucleophile. The hydrophobictunnel prevents protonation of NH3 and thus maintains its nucleophilicity.

As is the case in microorganisms, the α subunit canfunction in the absence of β subunit amidotransferaseactivity, provided ammonium is present at sufficientconcentration (Reaction 7.1).

However, the glutamine amidotransferase activity islikely of primaryimportance forAnS function inplants.

6. PAT, PAI and IGPS Enzyme Biochemical characterizations of PAT, PAI, and IGPS

lag behind molecular and genetic analyses. Phosphoribosylanthranilate transferase (PAT) is the

second enzyme in the tryptophan biosynthesis, andcatalyzes the formation of 5‐phosphoribosyl anthranilate(PRA) by transferring the phosphoribosyl moiety fromphosphoribosylpyrophosphate to anthranilate (Fig 7.25).

Phosphoribosylanthranilate isomerase (PAI), the thirdenzyme in the tryptophan biosynthesis, converts PRA to1-(o-carboxyphenyl-amino)-1-deoxyri-bulose5‐phosphate (CDRP) (Fig 7.27).

Indole‐3‐glycerol‐phosphate synthase (IGPS), thepenultimate enzyme of tryptophan biosynthesis,catalyzes the decarboxylation and ring closure of CDRP(Fig 7.28).

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Fig 7.25 Phosphoribosylanthranilatetransferase (PAT) catalyzes thesecond step of tryptophanbiosynthesis.

Fig 7. 27 Phosphoribosylanthranilateisomerase (PAI) catalyzes the thirdstep of tryptophan biosynthesis.

Fig 7.28 The indole-3-glycerol-phosphate synthase (IGPS)reaction produces the indolering found in tryptophan andsecondary metabolites.

A single‐copy gene encodes PAT in the Arabidopsisgenome.

PAT is constitutively expressed, and its mRNA levelis post‐transcriptionally enhanced by the first twointrons in Arabidopsis. PAT mutants fall into twogeneral classes: auxotrophic plants & prototrophs.

Auxotrophic plants have such low PAT enzymeactivity that seedlings cannot grow on sterile mediumwithout the addition of tryptophan.

Prototrophs have enough residual activity to survivewithout amino acid supply.

Adult trp1 auxotrophic mutants are small, bushy, andhave greatly reduced fertility, even when tryptophanis added to their growth medium (Fig 7.26).

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Fig 7.26 Morphology of thetryptophan‐requiring trp1mutants is drama-ticallyaltered from that of wild‐typeplants and includes smallstature and reduced apicaldominance. Source: Last,Cereon Genomics LLC,Cambridge, MA; previouslyunpublished.

The developmental defects could be caused by aninability to produce auxin (indole-3-acetic acid) or othermetabolites derived from this pathway.

Biochemical analysis of the prototrophic trp1 mutantsindicates the PAT enzyme activity is present in vastexcess of that required for maintaining pathway flux.

Five of the prototrophic mutants have 1% of wild‐typeenzyme activity or less, yet grow normally withouttryptophan supplementation.

In the case of PAI enzyme, Arabidopsis has three orfour highly homologous PAI genes depending onecotypes. In the Columbia ecotype, PAI1 and PAI2 are99% identical including their untranslated regions.

Owing to PAI gene redundancy, loss of any one genedoes not reduce enzyme activity sufficiently to producea distinct mutant phenotype.

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In the case of IGPS, it is the only enzyme known tocatalyze the formation of the indole ring.

One of two Arabidopsis genes encoding IGPS wasisolated based on its ability to complement an E. colitrpC-mutation despite low amino acid identity (22–28%)with microbial enzymes.

Plant IGPSs exist as monofunctional enzymes, incontrast to fungal and bacterial IGPSs, which aresynthesized as fusion proteins containing one or twoother enzymes of the tryptophan biosynthetic pathway.

Analysis of tryptophan and IAA levels in differentArabidopsis tryptophan biosynthesis mutants, as well asin transgenic plants with antisense suppression ofIGPS, suggested that indole‐3‐glycerol‐phosphate mightbe the branch‐point intermediate in tryptophan‐independent auxin biosynthesis.

7. Tryptophan synthase Tryptophan synthase (TS) is a bifunctional enzyme

that catalyzes the last two-step reaction of tryptophanbiosynthesis, the conversion of indole‐3‐glycerolphosphate (IGP) and serine to tryptophan (Fig 7.29).

TS, the best characterized of the microbial tryptophanbiosynthetic enzymes, exists as an α2β2 heterotetra-mer, and the separate subunits are capable ofcatalyzing independently two partial reactions of thereaction sequence:Reaction 1: TS α-subunitIGP indole + glyceraldehyde -phosphate [IGP lyase(IGL) activity]Reaction 2: TS β-subunitIndole + serine tryptophan + H2O

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Fig 7.29 Tryptophan synthase (TS)catalyzes the final step in tryptophanbiosynthesis.

Conversely, the rate of cleavage of IGP at theα‐subunit active site (Reaction 7.2) is increased20‐fold by binding of serine to the pyridoxal phosphatecofactor at the β‐subunit active site (Fig 7.30).

The two partial reac-tions are kept in phaseby allosteric interact-tions between the twosubunits, and eachsubunit can causeconformationalchanges that affect thecatalytic activity of theother.

For example, α‐subunit binding stimulates β activity(Reaction 2) 30‐fold compared with uncomplexed βsubunit.Fig 7.30 Tryptophansynthase (TS) consistsof α‐ and β subunits thatjoin to form two activesites with a hydrophobictunnel between themthat channels indole fromthe site where it isreleased from indole-3-glycerol phosphate to thesite where it iscondensed with serine.(A) The structure of TS from Salmonella typhimurium with α subunits inblue, β subunit N‐terminal residues in yellow, and C‐terminal residues inred. A molecule of indole‐3‐glycerol phosphate (red) is bound to theactive site of the α subunit. (B) Schematic drawing of the tunnel thatguides indole from the active site of the α subunit to that of the βsubunit. Source: (A) Hyde et al. (1998). J. Biol. Chem. 263:17857–17871.

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8. Biosynthesis in Plastids Aromatic amino acids are synthesized in the

chloroplast, and several lines of evidence show thatthe total set of enzymes for the biosynthesis ofphenylalanine, tryptophan, and tyrosine is found inplastids.

Isolated chloroplasts incorporate 14CO2 into aromaticamino acids, and activities of almost all enzymesinvolved in chorismate and aromatic amino acidbiosynthesis have been detected in chloroplastextracts.

Consistent with this view, genes containing plastidtargeting sequences have been cloned for at least oneisoenzyme responsible for a given biochemical step inthe plant aromatic amino acid pathway.

Plastid localization for most pathway enzymes wasconfirmed using GFP (green fluorescent protein)fusion proteins. Import of precursor proteins of DAHPsynthase, shikimate kinase, EPSP synthase, PAI, andTSA into isolated chloroplasts has been also beendemonstrated.- Subcellular fractionation and localization studies have

uncovered a cytosolic DAHP synthase activity in someplants, and cytosolic isoforms both of CM (probably all plants)and dehydroquinate dehydratase‐shikimate dehydroge-nase (so far only found in tobacco) as well.

- The last two enzymes were confirmed at both biochemical andgenetic levels. Genes encoding cytosolic tobacco (Nicotianatabacum) dehydroquinate dehydratase/shikimatedehydrogenase 2 and Arabidopsis and petunia (Petuniahybrida) CM2 have been identified, and the cytosoliclocalization of the corresponding proteins was demonstrated.

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9. Stress Conditions Enzymes of aromatic amino acid biosynthesis in plants

are inducible by environmental stress wounding andbacterial infection.

The kinetics of wound‐inducible DAHP synthase mRNAinduction are similar to that of phenylalanineammonia‐lyase (PAL), the committing enzyme ofaromatic secondary metabolism (Fig 7.31A).

Both mRNAs and proteins for all of the enzymes of thetryptophan pathway are induced after treatment withbacterial pathogens, and the rates of induction aresimilar to that for camalexin accumulation.

The timing of the responses is coordinated, and the amount ofcamalexin that accumulates is tightly correlated with thedegree of tryptophan pathway enzyme induction inresponse to various bacterial pathogens (Fig 7.31B).

(B) Infection with a bacterial pathogen increases mRNAs for Arabidopsistryptophan pathway enzymes and increases production of the antimicrobialindolic secondary metabolite camalexin, which fluoresces blue under UV light.

Fig 7.31 Plants induce theenzymes of aromatic aminoacid biosynthesis under con-ditions that cause increasesin aromatic secondary meta-bolism. (A) Wounding ofpotato tubers causesincreased DAHP synthasemRNA and enzyme activity.

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4. ASPARTATE-DERIVED AA1. Biosynthetic Pathways Three pathways lead directly from

aspartate to the amino acidslysine, threonine, and methionine(Figs. 7.2 and 7.32).

The current understanding is thatchloroplasts are fully autonomousin the biosynthesis of allaspartate-derived amino acids,including methionine (Fig 7.32).

Fig 7.32 The aspartate‐derived amino acids areproduced in the plastid. Methionine activation toS‐adenosylmethionine and methionineregeneration occur in the cytosol.

Aspartate‐derived amino acids are required in the diets ofnonruminant animals, including humans.

Some diets that rely on plant foods are deficient in one ormore of these protein building blocks and can lead tohealth problems. The amino acid deficiencies of grainsand legume seeds can be complemented by eating bothin combination.

Human vegetarians who do not eat any animal productsmust balance their diets with care to provide all essentialamino acids in the proportions that are needed for proteinsynthesis.

For example, corn has a poor amino acid content andcomposition, being especially deficient in lysine, but alsotryptophan and methionine. On the other hand, soybeanis lysine‐rich but methionine‐ and threonine‐poor.

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Aspartate provides the entire carbon skeleton forthreonine, and threonine biosynthesis seems to serveas the main pathway (Fig 7.33), with branches tolysine and methionine (Figs. 7.34 and 7.35).

Aspartate is activated by phosphorylation byaspartate kinase (AK; also called aspartokinase), thecommitting enzyme for all aspartate‐derived aminoacid biosynthesis.

Aspartate 4‐phosphate (β‐aspartyl phosphate) thengoes through two NADPH‐mediated reductions, whichare catalyzed sequentially by aspartate-semialdehy-de dehydrogenase and homoserine dehydrogena-se (HSDH), to yield homoserine.

Homoserine is then phosphorylated at the 4‐positionby homoserine kinase.

Fig 7.33 The threonine biosynthetic pathway

Homoserine 4‐ phosphate is at the branching pointof threonine and methionine biosynthesis.

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Fig 7.34 Lysinebiosynthesisbranches fromthe threoninepathway

In bacteria and plants, lysine is synthesized fromaspartate via the diaminopimelate (DAP) pathway (Fig7.34).

The committing enzyme in the DAP pathway of lysinebiosynthesis is dihydrodipicolinate synthase (DHPS),which condenses aspartate 4‐semialdehyde withpyruvate and cyclizes the product to 2,3‐dihydrodipi-colinate.

2. Sulfur Requirement Amino acid Methionine containing sulphur originates

from cysteine that acquires its carbon skeleton fromhomoserine carbon skeleton.

The activated form of homoserine in plants is homose-rine 4‐phosphate (O‐phosphohomoserine), with itscarbon skeleton coming originally from Aspartate,which is also the precursor of threonine (Fig 7.35).

In the trans‐sulfuration pathway, the thiol group ofcysteine is transferred to homoserine to producehomocysteine, through a cystathionine intermediate.

Cystathionine ‐synthase, another pyridoxal phos-phate-dependent enzyme, catalyzes the sulfur‐linkedjoining of cysteine and homoserine 4‐phosphate toform cystathionine (a thioether) and orthophosphate.

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Fig 7.35 The methionine biosynthetic pathway. THF, tetrahydrofolate

Next, the C3 skeleton of cysteine is cleaved bycystathionine β‐lyase (again pyridoxal phosphate-dependent), now leaving the sulfur atom attached tothe homoserine carbon skeleton; this produceshomocysteine, pyruvate, and ammonium, making thereaction essentially irreversible.

Direct sulfhydration of the activated homoserine withsulfide to give rise to homocysteine seems to be ofminor physiological significance in plants.

Homocysteine is then converted to methionine, withN5‐methyltetrahydrofolate serving as the methyldonor, in a reaction catalyzed by methionine synthase(homocysteine methylase).

This activity not only is involved in de novo methioninesynthesis, but also functions in recycling theS‐adenosyl‐L‐homocysteine produced when SAMdonates its methyl group in a methylation reaction (Fig7.36).

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Fig 7.36 S‐Adenosylmethonine is produced from methionine and can berecycled to regenerate methionine. THF, tetrahydrofolate

This activity not onlyis involved in denovo methioninesynthesis, but alsofunctions in recy-cling the S-adeno-syl-L-homocyste-ine produced whenSAM (S-adenosyl-methionine) dona-tes its methyl groupin a methylationreaction (Fig 7.36).

3. Biosynthetic Regulation The biochemical mechanisms regulating flux through

the branches of the aspartate‐derived amino acidpathway are complex.- First, aspartate‐derived metabolism can follow three major

routes, creating at least five branch point enzymes: aspartatekinase (AK), dihydrodipicolinate synthase (DHDPS),homoserine dehydrogenase (HSDS), threonine synthase (TS),and cystathionine ‐synthase (CS). Of these, the first four areallosterically regulated in plants, and CS is regulated at thetranscriptional and, in some plants, at the posttranscriptionallevel.

- Second, the products of these pathways are expected to beneeded by the plant in different amounts at each stage ofdevelopment.

An overview of the types of regulatory mechanismsinferred by in vitro studies is shown in Figure 7.37.

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Fig 7.37 Biochemical regulatory mechanisms proposed to regulate thesynthesis of amino acids derived from aspartate. Red lines indicateend‐product inhibition, the purple line refers to a reduction in detectableenzyme, and the green lines indicate stimulation of activity

AK, the committingenzyme for theoverall pathway, is acritical regulatoryenzyme occurring inmultiple classes ofAK isoenzymes,which have divergentprimary sequencesand contrastingallosteric properties.

If feedback regulation of AK is primarily responsible forregulating flux to all three end products (threonine,lysine, and methionine/SAM), deregulating this enzymeshould increase accumulation of all three amino acids,and this is indeed the case.

Mutants with deregulated lysine‐sensitive AK wereselected by taking advantage of the fact thatadministration of threonine and lysine causes starvationfor methionine by reducing total AK activity throughfeedback inhibition.

Mutants were selected with AK activities that were lesssensitive to lysine inhibition (Fig 7.38).

Wild‐type plants or plant cell cultures on mediumcontaining a mixture of lysine and threonine were deathby methionine starvation due to inhibition of AK activity(Fig 7.38).

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Fig 7.38 Treatment of plants with lysine plus threonine allows selection forfeedback‐insensitive aspartate kinase mutants. AKLys and AKThr refer to AKisoforms that are inhibited by lysine or threonine, respectively. (A) Growth ofwild‐type plants or plant cell cultures on medium containing a mixture oflysine and threonine causes death by methionine starvation because ofinhibition of AK activity. (B) Mutants arise that are insensitive to this normallytoxic amino acid mixture. These plants (AK*Lys ) contain amino acid changesin their lysine‐sensitive aspartate kinase activities, which make theminsensitive to feedback inhibition. This restores the plant’s ability tosynthesize methionine.

In plants, DHDPS activity is very sensitive to lysineinhibition (the concentration to achieve 50% inhibition,or I50, is 10–50 μM), making it a likely candidate for akey regulator of lysine accumulation.

Plant mutants with desensitized DHDPS were identi-fied in several species by selection for resistance tothe toxic lysine analog S‐aminoethyl‐l‐cysteine (AEC).

This molecule competes with lysine for incorporationinto proteins and allows the identification oflysine‐overproducer plants.

AEC resistance in Nicotiana sylvestris, caused byproduction of a lysine‐insensitive mutant DHDPSenzyme with a single amino acid change, led to theseplants accumulating about 10‐fold more lysine thandid the wild type (Fig 7.39).

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FIGURE 7.39 Dihydrodipicolinate synthase (DHDPS) regulates theaccumulation of lysine in plants. (A) Feedback inhibition of DHDPS acts tocontrol the flux from aspartate 4‐semialdehyde into lysine. (B) Dominantfeedback‐insensitive DHDPS mutants can be selected by use of classicalgenetics (DHDPS* AEC) or by expression of feedback‐insensitive E. colienzyme in transgenic plants (DHDPS E. coli). These plants overproducelysine in comparison with the plant that expresses the wild‐type DHDPSactivity

4. Lysine Biosynthesis and Degradation Plants expressing feedback‐insensitive mutant

DHDPS accumulate high quantities of lysine, affectingother AA, causes severe developmental defects.

Increasing lysine biosynthesis by simply expressingmutant DHPS in a seed‐specific manner may not be aapt way to increase the nutritional value of crops thatrequires engineering increased lysine content inseeds.

Lysine degradation in a concerted manner withbiosynthesis is necessary to maintain a certain level oflysine. The first enzyme of lysine catabolism is lysine-ketoglutarate reductase (LKR) which reduces thelabile Schiff base, formed between the ε‐amino groupof lysine and α‐ketoglutarate, to a stable opine,saccharopine (Fig 7.40).

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FIGURE 7.40 Initial reactions in lysine catabolism

In the subsequent reaction, catalyzed by saccharopinedehydrogenase (SDH), the amine function istransferred to the α‐ketoglutarate skeleton, giving riseto glutamate while the ε‐C of the lysine C‐skeletonnow carries the carbonyl function (α‐aminoadipicδ‐semialdehyde, which is further degraded to yieldacetyl‐CoA).

5. BRANCHED-CHAIN AA Branched‐chain amino acids are AA having small

branched hydrocarbon residues and consist ofIsoleucine, leucine, and valine.

The biosynthesis of the branched‐chain amino acids byplants attracts considerable attention for severalreasons:- First, they represent three of the 10 essential amino

acids that animals must obtain from their diet and are,therefore, nutritionally important.

- Second, this pathway is a significant target for avariety of commercially successful herbicides.

- Finally, the amino acid products serve as precursorsfor secondary metabolites (Fig 7.41).

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Fig 7.41 The branched-chain amino acids serve asprecursors to plantsecondary metabolites.

1. Threonine Deaminase The biosynthesis of isoleucine,

begins with deamination ofthreonine, while leucine andvaline begin with the reaction ofPyruvate and hydroxyethyl-TPP(Fig. 7.41).

A unique feature of branched-chain amino acid biosynthesis isthat isoleucine and valine aresynthesized in chloroplasts fromtwo parallel pathways utilizing thesame set of four enzymes(Fig.7.42).

Each of the four enzymeshas dual substrate specifi-city, starting with eitherpyruvate (leading tovaline) or α‐ketobutyrate(leading to isoleucine).

While pyruvate (C3) is anintermediate of glycolysis,its C4 counterpart, α‐keto-butyrate, is produced bythreonine deaminase (TD,also known as threoninedehydratase).

TD is thus the committingenzyme of the isoleucinepathway and has no role invaline metabolism.

Fig 7.42 Biosynthesis of isoleucine,leucine, and valine

1

2

3

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2. AHAS and IPMS The first common enzyme in the biosynthesis of the

branched-chain amino acids is acetohydroxyacidsynthase (AHAS; also known as acetolactatesynthase), which catalyzes the decarboxylation ofpyruvate followed by its condensation with eitherpyruvate or 2‐ketobutyrate to form 2‐acetolactate or2‐acetohydroxybutyrate, respectively (Fig 7.42).

AHAS requires thiamine diphosphate as cofactor,which is anchored to the active site of the enzyme bya divalent metal ion such as Mg2

+. AHAS also requires flavin adenine dinucleotide (FAD),

even though this cofactor does not participate in theprincipal reactions and may have instead a structuralfunction.

AHAS has been intensely studied because it is thetarget of five commercially important and structurallyhighly divergent classes of herbicides: imidazolino-nes, sulfonylureas, triazolopyrimidines, pyrimidyl-oxy‐benzoates and sulfonylaminocarbonyltriazoli-nones (Fig 7.43).

Leucine biosynthesis branches from 2‐ketoisovale-rate, the last intermediate of the valine biosyntheticpathway, and evidence indicates that plants use thepathway found in microbes (Fig 7.42).

The committing enzyme for the pathway, 2‐isopropyl-malate synthase (IPMS), catalyzes the transfer of anacetyl group from acetyl‐CoA to 2‐ketoisovalerate toproduce 3‐carboxy‐3‐hydroxyisocaproate (2‐isopropyl-malate or α‐isopropylmalate).

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Fig 7.43 Examples of the fiveclasses of commercialherbicides that are AHASinhibitors.

6. GLUTAMATE-DERIVED AA Proline, arginine, and the (nonproteinogenic) amino acid

ornithine are biosynthetically derived from glutamate. Proline has received major attention in plant biology

because of its accumulation in responses to salt,drought, and metal stress.

Although biochemical studies have rather neglected thesynthesis of arginine in plants in the past, genomicannotations have provided missing information.

1. Proline Metabolism Understanding and manipulating proline biosynthesis

in plants is largely motivated by observations that thiscyclic secondary amino acid is one of several organiccompounds that can function as compatible solutes.

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- Compatible solutes are molecules that can accumulate to highconcentrations in the cytoplasm without disrupting cellularactivity and allow plants to lower their water potential andmaintain turgor during dry or saline conditions.

Proline also scavenges reactive oxygen species(ROS), which are formed under stressful conditions,and its function in stress situations is thusmultifaceted.

Studies of genetically engineered plants, accumulatinghigh levels of proline, have provided clear evidence forthe ability of proline to confer increased osmotictolerance.

Proline can be synthesized by two different pathwaysin plants: the first originates from glutamate, thesecond from ornithine, although the details of theornithine pathway are less clear (Fig 7.44).

In plants, the γ‐glutamyl kinase (proB in E. coli) and GSA dehydrogenase(proA in E. coli) are synthesized in a protein fusion. This activity is referred toas Δ1‐pyrroline‐5‐carboxylate synthetase.

Fig 7.44 Multiple path-ways are proposed forproline biosynthesis inplants. A glutamate-derived pathway is wellestablished (top-down),and increasingevidence suggests theimportance of ornithineas a precursor. Therelative importance ofthe pathways throughornithine‐δ‐aminotransferase or ornithine-α-aminotransferaseremains to bedetermined.

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The committing enzyme of the glutamate pathway,pyrroline‐5‐carboxylate synthetase (P5CS), is abifunctional enzyme in plants.

2. Metabolism Regulation Regulation of proline accumulation in plants occurs

both at the enzyme level and through changes in geneexpression.

The committing enzyme P5CS appears to be ratelimiting in plants, and its transcript as well as freeproline increase rapidly in response to desiccation,salt stress and ABA (abscisic acid) treatment, andthey return to normal level when dehydrated plantsare watered.

In several plant species, PC5S overexpression (butnot that of P5C reductase) leads to plants withincreased soluble proline content and reducedsensitivity to osmotic stress.

As the γ‐glutamyl kinase activity of PC5S isfeedback‐inhibited by proline, this feedback loopparadoxically appears to counteract the effect ofincreased expression of the enzyme.

Of the two PC5S genes of Arabidopsis, PC5S1responds to osmotic stress, and the loss‐of‐functionmutant displays increased ROS levels andhypersensitivity to stress.

Mutant pc5s2 plants, on the other hand, havedevelopmental defects, in particular embryo abortionduring late seed development (desiccation stage).

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3. Arginine Biosynthesis Arginine has a high N:C ratio (4:6) and serves as

nitrogen storage compound in seeds, either in freeform (e.g., in pea seeds) or in protein‐bound form.

In addition, it serves as a precursor in the biosynthesisof polyamines, certain alkaloids, and the signalmolecule nitric oxide (NO).

In terrestrial (ureotelic) vertebrates, it is the immediateprecursor of urea, the nitrogen excretion product inurine.

A limited number of biochemical and in silico genomemining studies have allowed elucidation of thepathway of arginine biosynthesis in plants, and thesereactions are more or less identical to those in otherorganisms (Fig 7.45).

The pathway can be divided into two parts: fromglutamate to ornithine, and from ornithine to arginine.

The ornithine pathwaybegins with theN‐acetylation ofglutamate byN‐acetylglutamatesynthase (NAGS).

Fig 7.45 Biosynthesis ofarginine. Structures are onlyshown for the pathway fromornithine to arginine. Structuresin the ornithine pathwaycorrespond to those also seenin Figure 7.42, with the notabledifference that they areN‐acetylated.

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The following ‐phosphorylation and subsequent reductionto the semialdehyde are analogous to the reactions of freeglutamate in proline biosynthesis.- Because acetylation of the amino group prevents spontaneous

cyclization to the pyrroline carboxylate, N‐acetylglutamicsemialdehyde can be transaminated to give rise toN‐acetylornithine, from which ornithine is released either byhydrolysis or by transfer of the acetyl moiety to glutamate(transacetylation) within the cyclic pathway (Fig 7.45).

Arginine synthesis is initiated by the carbamoylation of theδ‐amino group of ornithine with carbamoyl phosphate asthe activated intermediate in a reaction catalyzed byornithine carbamoyl transferase (OCT) that producescitrulline (Fig 7.45).

The N atom required to convert citrulline to arginine istransferred from aspartate via argininosuccinate, which iscleaved to yield arginine and fumarate.

7. HISTIDINE Histidine (His), one of the standard amino acids in

proteins and plays a critical role in plant growth anddevelopment, was discovered (L-histidine) independentlyby Kossel and Hedin in 1896.

Uniquely among the twenty standard amino acids, theimidazole side group of His has a pKa of approximately6, allowing it to alternate between the protonated andunprotonated states under physiological conditions.

The imidazole side group is a weak acid with a pKa of approximately 6,allowing it to switch between the protonated and unprotonated statesunder cellular conditions.

Fig 1. L-histidine structure.

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His biosynthesis in plants, occuring via the samemetabolic route as in micro-organisms, begins with thecondensation of 5′-phosphoribosyl 1-pyrophosphate(PRPP) and ATP (Fig. 2).

Plant genes encoding all of the eight enzymes requiredfor His synthesis have been identified.

Release of pyrophosphate by hydrolysis produces5′‐phosphoribosyl AMP. The six‐membered ring of AMPopens by hydrolysis followed by the isomerization ofketone.

Ammonia provided by hydrolysis of glutamine, in aglutamine amidotransferase reaction and being guidedthrough a tunnel, affects the production of imidazoleglycerol phosphate (IGP) and 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR).

Fig 2. The histidine biosyntheticpathway in Arabidopsis. Abbrevia-tions of enzyme names are indicatedin parentheses, and the correspon-ding Arabidopsis gene names andAGI codes are shown in blue.Allosteric inhibition of ATP-PRTactivity by L-His is indicated in red.PRPP (5′-phosphoribosyl-1-pyrophos-phate), PRATP (N′-5′-phosphoribosyl-ATP), PRAMP (N′-5′-phosphoribosyl-AMP), ProFAR (N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxa-mide) ribonucleotide, PRFAR (N′-[(5-phos-phoribulosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide, IGP (imi-dazole glycerol-phosphate), IAP (imida-zole acetol-phosphate), AICAR (5′-phos-phoribosyl-4-carboximide-5-aminoimida-zole) and 2-OG (2-oxoglutarate).Hyperlinks to chemical structures andTAIR locus pages are provided

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Fig 7.46 Biosynthesisof histidine

AICAR is used in a salvage pathway to regenerate ATP,while IGP is converted by consecutive dehydration,transamination, hydrolysis of the phosphate group, andtwo dehydrogenation steps to histidine (Fig 7.46)

All proteins involved in the histidine pathway carry anN‐terminal extension either proven or assumed to directthe proteins to the plastids.

It is generally accepted that the complete pathwayoperates in the plastid.

His is the fourth most metabolically expensive aminoacid to synthesize (after the aromatic amino acids Phe,Trp and Tyr), with estimates of 31- 41 ATP moleculesrequired per molecule of His produced.

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SUMMARY1. Amino acids, the building blocks of proteins in all organisms, play

additional roles in plants.2. In plants, amino acids serve as precursors to a plethora of natural

products that provide defense against pathogens and herbivoresand tolerance to abiotic stress.

3. They also store or transport nitrogen from sources to sinks. Thecontrol of amino acid synthesis in plants, therefore, affects manyaspects of growth, development, and survival.

4. The synthesis of essential amino acids in plants and the amino acidcomposition of seeds relate indirectly to animal and human nutrition.

5. Thus, understanding the pathways that allow and control amino acidsynthesis in plants has significance with regard to basic research onthe control of metabolic pathways as well as practical implications.

6. Although amino acid biosynthetic pathways have been well definedin microbes, the situation in plants is still less defined, in partbecause of additional unique complexities.

7. For example, in many instances, plants have multiple isoenzymesthat catalyze the same biosynthetic reactions. These isoenzymesmay be localized in distinct organelles or distinct cell types, or maybe present at different developmental stages.

8. Defining each step in an amino acid biosynthetic pathway anddetermining how each step is regulated, not only within the contextof the respective pathway but rather in the context of the generalmetabolic network, are some of the key aspects of current researchin amino acid biosynthesis in plants.

9. Molecular, genetic, and biochemical approaches have beencombined to elucidate the steps of these pathways in plants and tounderstand the regulation of these pathways at the level of generegulation and beyond.

10. Plant mutants in amino acid biosynthetic enzymes, which can nowreadily be identified in T‐DNA insertion lines, have shown that thesynthesis of amino acids in vivo affects numerous diverseprocesses, including photorespiration, hormone biosynthesis, andplant development.

11. Thus, while being products of primary metabolism, amino acids alsocontrol many diverse aspects of plant growth and development.

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5. Research of AA Pathways Uncovering the features of amino acid biosynthesis

unique to plants should stimulate both fundamentaland applied research aimed at understanding;- the genes that control growth‐limiting processes (e.g., the

assimilation of inorganic nitrogen into amino acids) and- the factors that regulate the synthesis of plant secondary

compounds from amino acid precursors.

Such studies should also provide a framework formanipulating amino acid biosynthesis pathways intransgenic plants.

For example, enzymes in several pathways havebeen identified as targets for herbicides, and in somecases, the genes encoding these enzymes havebeen used for engineering herbicide resistance (Box7.1).