Biosynthesis of food constituents: Amino acids: 2 The alanine valine leucine, serine cysteine glycine, and aromatic and heterocyclic amino acids groups – a review

1 THE ALANINE, VALINE, AND LEUCINE GROUP 1.1 Alanine

L-Alanine and the essential amino acids L-valine and L-leucine have a common precursor, i.e. pyruvic acid from the glycolytic pathway (Figure 1).

Biosynthesis of Food Constituents: Amino Acids:

2. The Alanine-Valine-Leucine, Serine-Cysteine-Glycine, and

Aromatic and Heterocyclic Amino Acids Groups – a Review

JAN VELÍŠEK and KAREL CEJPEK

Department of Food Chemistry and Analysis, Faculty of Food and Biochemical

Technology, Institute of Chemical Technology Prague, Prague, Czech Republic

Abstract

VELÍŠEK J., CEJPEK K. (2006): Biosynthesis of food constituents: Amino acids: 2. The alanine-valine-leucine, serine-cysteine-glycine, and aromatic and heterocyclic amino acids groups – a review.Czech J. Food Sci., 24: 45–58.

This review article gives a survey of principal pathways that lead to the biosynthesis of the proteinogenic amino acids of the alanine-valine-leucine group starting with pyruvic acid from the glycolytic pathway and serine-cysteine-glycine group starting with 3-phospho-D-glyceric acid from the glycolytic pathway. A survey is further given to the aromatic and heterocyclic amino acids (phenylalanine, tyrosine, tryptophan, histidine) starting with 3-phosphoenolpyruvic acid from the glycolytic pathway and D-erythrose 4-phosphate, an intermediate in the pentose phosphate cycle and Calvin cycle.

Keywords: biosynthesis; amino acids; alanine; valine; leucine; serine; glycine; cysteine; phenylalanine; tyrosine; tryp-tophan; histidine

Partly supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project MSM 6046137305.

Alanine is formed from pyruvic acid in a simple transamination reaction (Figure 2) catalysed by alanine transaminase (EC 2.6.1.2) (SENGBUSCH). The transamination reactions are catalysed by transaminases dependent on the coenzyme pyri-doxal 5'-phosphate (PLP).

H3C COOH

O

L-glutamic acid

EC 2.6.1.2

CH3 COOH

NH2

2-oxoglutaric acid

pyruvic acid L-alanine

Figure 1 Figure 2

pyruvic acid Glycolysis

L-valine

46

1.2 Valine

Valine is synthesised in a set of four reactions, and an extension of the valine pathway results in leucine synthesis (Figure 3) (IUBMB 2003). The first three enzymes involved in the biosynthesis of valine and leucine, i.e. acetolactate synthase (EC 2.2.1.6), ketol-acid reductoisomerase (EC 1.1.1.86), and di-hydroxyacid-dehydratase (EC 4.2.1.9), also catalyse analogous reactions leading to the biosynthesis of isoleucine. 3-Methyl-2-oxobutanoic acid is the common precursor of both amino acids, valine and leucine. This carboxylic acid then yields valine by the action of the branched-chain-amino-acid transaminase (EC 2.6.1.42).

1.3 Leucine

Leucine is synthesised from 3-methyl-2-oxobu-tanoic acid in six steps (Figure 3) (IUBMB 2003). The first step, catalysed by 2-isopropylmalate

synthase (EC 2.3.3.13, requires K+ _{ions), is the} condensation of 3-methyl-2-oxobutanoic acid with acetyl-CoA to (S)-2-isopropylmalic acid. The next two steps are catalysed by 3-isopropylmalate dehydratase (EC 4.2.1.33). 2-Isopropylmaleic acid arises from 2-isopropylmalic acid by elimination of water and addition of water to 2-isopropyl-maleic acid yields (2R,3S)-3-isopropylmalic acid. The enzyme also hydrates the product back to (S)-2-isopropylmalic acid, thus bringing about the interconversion between the two isomers. In the next step, 3-isopropylmalic acid is oxi-dised to (S)-2-isopropyl-3-oxosuccinic acid by the NAD+_{-dependent 3-isopropylmalate} dehydroge-nase (EC 1.1.1.85). The product decarboxylates spontaneously to 4-methyl-2-oxopentanoic acid (4-oxoisocapronic acid), which is, in the last step, transformed to leucine by either branched-chain-amino-acid transaminase (EC 2.6.1.42) or more specific leucine transaminase (EC 2.6.1.6) that does not act on valine or isoleucine.

Figure 3

EC 2.6.1.6

OH

L-glutamic acid

H3C COOH

CH3 COOH

H3C COOH

CH3 COOH

OH

H3C COOH

CH3 COOH

O

H3C COOH

CH3 O

H3C COOH

CH3 NH2

EC 2.2.1.6

EC 4.2.1.9

H3C

O

COOH CH3

OH CO2

H2O

H3C COOH

CH3

O

H3C COOH

CH3

OH

(S)-2-hydroxy-2-methyl-3-oxobutanoic acid (R)-2,3-dihydroxy-3-methylbutanoic acid

3-methyl-2-oxobutanoic acid pyruvic acid

H3C COOH

O 2

EC 1.1.1.86

NADPH + H NADP

H3C COOH

CH3 COOH

OH

H3C S

O

CoA

HS CoA + H2O

H2O

H2O

NAD NADH + H CO2 2-oxoglutaric acid

(S)-2-isopropylmalic acid 2-isopropylmaleic acid

(2R,3S)-3-isopropylmalic acid (S)-2-isopropyl-3-oxosuccinic acid 4-methyl-2-oxopentanoic acid L-leucine

H3C COOH

CH3

NH2

L-valine

EC 2.3.3.13 EC 4.2.1.33

EC 4.2.1.33

EC 1.1.1.85 EC 2.6.1.42

EC 2.6.1.42

2 THE SERINE, GLYCINE, AND CYSTEINE GROUP

2.1 Serine

L-Serine is generated in a three-step reaction from 3-phospho-D-glyceric acid, formed in the glycolytic pathway, and becomes the precursor of glycine and L-cysteine (Figure 4).

2.2 Glycine

The major pathway of glycine biosynthesis is the direct transformation of L-serine, which is catalysed by PLP protein, i.e. serine hydroxymethyl trans-ferase (EC 2.1.2.1). The hydroxymethyl group of the serine side chain is split off and this C1 unit is ac-cepted by tetrahydrofolic acid (H₄PteGlu, R = ben- zoyl glutamic acid residue) under the formation of 5,10-methylene-tetrahydrofolic acid (5,10-meth-ylene-H₄PteGlu) (Figure 6) (SENGBUSCH).

2.3 Cysteine

Cysteine directly or indirectly provides sulfide for structural, regulatory, or catalytic purposes in peptides, proteins, and numerous low molecular weight compounds such as methionine, gluta- thione, biotin, and thiamine. Biosynthesis of the cysteine -SH group needs sulfur and thus provides an exclusive entry of reduced sulfur into cellular metabolism. Plants take up sulfur as inorganic sulfate ions. After its uptake by roots, sulfate is dis-tributed into different organs and, to be assimilated, it has to be reduced in a process called assimila-tory sulfate reduction performed in chloroplasts. The first step in the sulfate assimilation pathway is catalysed by ATP sulfurylase (EC 2.7.7.4). This enzyme activates sulfate via an ATP-dependent reaction that leads to the formation of adenylyl-The first step of serine biosynthesis is the

oxida-tion of 3-phospho-D-glyceric acid at C-2 to 3-phos-phopyruvic acid catalysed by 3-phosphoglycerate dehydrogenase (EC 1.1.1.95). This phosphorylated oxo-analogue of serine is transaminated by PLP-de-pendent phosphoserine transaminase (EC 2.6.1.52) in the second step yielding 3-phospho-L-serine. Finally, in the third step, phosphoserine is hydro-lysed by phosphoserine phosfatase (EC 3.1.3.3) to serine (Figure 5) (SENGBUSCH).

Figure 4

Figure 5

Figure 6

3-phospho-D-glyceric acid

L-serine L-cysteine

glycine Glycolysis

L-glutamic acid

PO COOH OH

PO COOH O

PO COOH

NH2

HO COOH

NH2

2-oxoglutaric acid NAD NADH + H

EC 1.1.1.95 EC 2.6.1.52 EC 3.1.3.3

3-phospho-D-glyceric acid 3-phosphopyruvic acid 3-phospho-L-serine L-serine

P H2O

NH2 COOH

HO COOH + H2O

NH2 _{EC 2.1.2.1}

L-serine H4PteGlu glycine 5,10-methylene-H4PteGlu H

N

N N

N OH

H2N

N R

H H

5 10

N

N N

N OH

H2N

N R H

48

sulfate (adenosine-5'-phosphosulfuric acid, APS) and inorganic diphosforic acid (PP).

To accomplish the incorporation of sulphur into biomolecules, specifically amino acids, sulphate in APS is transformed into sulphite and this into sulphide. This process may occur through two different pathways, depending on the organism. One of them involves phosforylation of APS by adenosine 5'-phosphosulfate kinase (EC 2.7.1.25) using ATP to produce 3'-phosfoadenosine-5'-phos- phosulfate (PAPS) and ADP. In the following re-action, PAPS reductase (EC 1.8.4.8) firstly reacts with reduced thioredoxin and then with PAPS to generate free sulphite. The other pathway involves the direct reduction of APS by 5'-adenylylsulfate reductase (EC 1.8.4.9), which uses GSH as an electron source to produce sulphite. In yeasts and many bacteria, sulphite is synthesised via adeno-sine 5'-phosphosulfate kinase, whereas in plants, green algae, and phototrophic bacteria, sulfate is transformed into sulphite via 5'-adenylylsulfate reductase (Figure 7).

Once sulfate has been reduced to sulphite, the subsequent step is identical in bacteria, fungi,

and plants. Sulfite is reduced to sulphide at the expense of oxidising three molecules of NADPH, by sulphite reductase (EC 1.8.7.1)1_.

Depending on the organism, there are two dif-ferent ways by which sulfide is incorporated into a carbon backbone to produce L-cysteine (Figure 8). First, the physiological O-ester substrate O -ace-tylserine is synthesised from L-serine by serine acetyltransferase (EC 2.3.1.30). Sulfide is condensed with O-acetylserine by the PLP-dependent enzyme O-acetylserine (thiol)-lyase (also called O -acetyl-serine sulfhydrylase, EC 2.5.1.47) to form cysteine directly. Because the substrate O-acetylserine is derived from the carbon and nitrogen assimilatory pathways, the O-acetylserine (thiol)-lyase links the sulfur and nitrogen assimilatory pathways together.

O-acetylserine sulphhydrylase (EC 2.5.1.47) also catalyses the condensation of sulfide with O -acetyl-homoserine to form L-homocysteine. O -Acetyl-homoserine is synthesised from L--Acetyl-homoserine by homoserine O-acetyltransferase (EC 2.3.1.31). Then homocysteine is transformed into cysteine by trans-sulfuration, i.e. L-homocysteine

asso-1_{Sulphite reductase contains a special acidic heme group called siroheme and catalyses the reduction of sulfite using}

electrons donated by ferredoxin.

Figure 7

N N N

N NH2

CH2

OH OH

O

adenosine-5´-phosphate

N N N

N NH2

CH2

OH OH

O O P O

O OH

S OH

O O

adenosine-5´-phosphosulfate

HO SH HS

EC 1.8.4.8

thioredoxin (oxidized) thioredoxin (reduced)

O S OH OH

sulfurous acid adenosine-5´-phosphosulfate-3´-phosphate

N N N

N NH2

CH2

OH O O P O

O OH

S OH

O O

O P OH

OH O

N N N

N NH2

CH2

OH O P O O OH

O P OH

OH O

adenosine-3´,5´-diphosphate

N S R

O

S _O

R H

N R

O H

R O

EC 2.7.1.25

ATP

ADP

O S OH OH

sulfurous acid

P O O OH

HO

+

EC 1.8.4.9 2 GSH G-S-S-G

ciates with serine to form L,L-cystathionine by action of cystathionine β-synthase (EC 4.3.1.22). Cystathionine in turn dissociates into cysteine, 2-oxobutyric acid, and ammonia by cystathionine γ-lyase (EC 4.4.1.1) (Figure 9) (JOST et al. 2000). Cysteine may also be transformed into homo-cysteine by reverse trans-sulphuration catalysed by cystathionine γ-synthase (EC 2.5.1.48) and cystathionine β-lyase (EC 4.4.1.8).

The serine acetyltransferase pathway is used by plants and by enteric bacteria. Fungi use different cysteine biosynthetic pathway depending on the species. In plants, cysteine can also be produced by the degradation of methionine.

3 THE AROMATIC AND HETEROCYCLIC AMINO ACIDS GROUP

3.1 Phenylalanine and tyrosine

Biosynthetic pathways of the aromatic and het-erocyclic amino acids family is schematically shown in Figure 10. The shikimic acid pathway provides a

route to the aromatic amino acids L-phenylalanine and L-tyrosine and to the heterocyclic amino acid L-tryptophan (IUBMB 2001). Shikimic acid2_{, a} central intermediate in this pathway, is formed by a sequence of reactions from 3-phospho- enolpyruvic acid (from glycolysis) and D-eryth-rose 4-phosphate (from the pentose phosphate cycle and the Calvin cycle). Both shikimic acid and the subsequent chorismic acid are important intermediates. The latter is the starting compound for three different pathways that lead to the end products phenylalanine, tyrosine, and tryptophan. An activated form of D-ribose 5-phosphate (5-phospho-α-D-ribose 1-diphosphate), is an-other intermediate of tryptophan biosynthesis. It has also a key position in the biosynthesis of L-histidine. The biosynthetic pathway of histidine is unusual in that histidine is produced from a purine. Animals employ none of these pathways and, accordingly, these amino acids feature among those essential amino acids for man that have to be obtained in the diet.

2_{Shikimic acid has been isolated from plants of Illicium anisatum L., syn. I. religiosum Sieb. et Zucc., Japanese shikimi.}

Figure 9 Figure 8

HO COOH

NH2

L-serine

O COOH H3C

O

NH2

HS COOH

NH2

acetyl-CoA HS-CoA _H

2S CH3 COOH

O-acetyl-L-serine L-cysteine

EC 2.3.1.30 EC 2.5.1.47

COOH HO

NH2

L-homoserine

COOH

NH2

COOH

S HOOC

NH2

NH2

acetyl-CoA HS-CoA

L-cysteine L,L-cystathionine

EC 2..3.1.31

H3C O COOH

O NH2

H2S CH3 COOH

L-serine

H2O

H3C

COOH O + NH3

O-acetyl-L-homoserine

2-oxobutyric acid

EC 2.5.1.47

EC 4.2.1.22

EC 4.4.1.1

COOH HS

NH2

L-homocysteine

50

The shikimic acid pathway begins with an aldol-type condensation of phosphoenolpyruvic acid with D-erythrose 4-phosphate to give the seven-carbon 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (3-deoxy-7-phospho-D-arabino-heptulosonic acid, Figure 11). This reaction catalysed by 3-deoxy-7-phosphoheptulonate synthase (EC 2.5.1.54) is mechanistically a complex reaction (Figure 12) (IUBMB 2002). Elimination of phosphoric acid from 3-deoxy-7-phospho-D-arabino-heptulosonic acid followed by intramolecular aldol reaction generates the first alicyclic intermediate 3-dehyd-roquinic acid. The elimination of phosphoric acid by 3-dehydroquinate synthase (EC 4.2.3.4) actually follows a NAD+_{-dependent oxidation (NAD}+ _is bound as a prosthetic group) of the central sec-ondary hydroxyl group, which is then re-formed in a NADH-dependent reduction reaction on the intermediate carbonyl compound prior to the aldol reaction occurring (Figure 13) (BENDER et al. 1989; GOURLEY et al. 1999; IUBMB 2001).

Reduction of 3-dehydroquinic acid with either quinate dehydrogenase (EC 1.1.1.24) or quinate/shi-

kimate dehydrogenase (EC 1.1.1.282) yields quinic acid, a fairly common acid frequently occurring in foods in the free and esterified form3_. 3-Dehy-droshikimic acid is formed from 3-dehydroquinic acid by dehydration catalysed by 3-dehydroquin-ate dehydratase (EC 4.2.1.10) (Figure 14) (IUBMB 2001). Reduction of 3-dehydroshikimic acid by NADP+_{-dependent shikimate 3-dehydrogenase} (EC 1.1.1.25) or quinate/shikimate dehydrogenase (EC 1.1.1.282) yields shikimic acid. This second shikimate dehydrogenase enzyme differs from shikimate 3-dehydrogenase (EC 1.1.1.25) in that it can use both quinate and shikimate as substrate and either NAD+ _{or NADP}+ _{as acceptor.}

An important branch point compound in the shikimate pathway is chorismic acid. Chorismic acid forms from shikimic acid, which is first phos-phorylated by shikimate kinase (EC 2.7.1.71) and yields 3-phosphoshikimic acid. 3-Phosphoshikimic acid reacts, under catalysis of 3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19), with fur-ther molecule of phosphoenolpyruvic acid, which is incorporated as an enol ether side-chain in 3_{Reduction of 3-dehydroquinic acid in bacteria can be achieved also by a quinoprotein, NAD(P)-independent}

quinate-dehydrogenase (pyrroloquinoline-quinone) (EC 1.1.99.25).

Figure 10

10

Glycolysis

Photosynthesis Pentose phosphate cycle

3-phosphoenolpyruvic acid

shikimic acid

chorismic acid

prephenic acid

L-phenylalanine L-tyrosine

L-tryptophan

L-histidine D-ribose 5-phosphate D-erythrose 4-phosphate

EC 2.5.1.54

EC 4.2.1.10

EC 1.1.1.25 EC 1.1.1.282 EC 2.7.1.71

EC 2.5.1.19

EC 4.2.3.5

EC 4.2.3.4 EC 1.1.1.24 EC 1.1.1.282

5-(1-carboxyvinyl)-3-phosphoshikimic acid chorismic acid

CH=O

H OH

H OH

CH2OP

H2C COOH

OP H2O

P

COOH

CH2

CH2OP

HO H

H OH

H OH

O P

OH

HO COOH

OH O

OH

HO COOH

OH HO

OH OH COOH

O OH

OH COOH

HO OH

OH COOH

PO

OH O COOH

PO COOH

CH₂ H2C COOH

OP

OH O COOH

COOH CH2

phosphoenolpyruvic acid

D-erythrose 4-phosphate +

3-deoxy-7-phospho-D-arabino

-heptulosonic acid 3-dehydroquinic acid quinic acid

3-dehydroshikimic acid shikimic acid

H₂O

NADPH + H NADP

NADP NADPH + H ATP

ADP

3-phosphoshikimic acid

phosphoenolpyruvic acid

P P

CH=O H OH H OH CH2OP

H2C COOH

OP

COOH CH2

CH2OP

HO H H OH H OH O

phosphoenolpyruvic acid

D-erythrose 4-phosphate

3-deoxy-7-phospho-D-arabino-heptulosonic acid

enzyme _O

O H

H

P _H

2O

enzyme _O

O

CH2 COOH

O P

H

O O

COOH H2C

enzyme

Figure 11

52

5-(1-carboxyvinyl)-3-phosphoshikimic acid (Fig-ure 15). The transformation of 5-(1-carboxyvinyl)-3-phosphoshikimic acid to chorismic acid involves a 1,4-elimination of phosphoric acid by chorismate synthase (EC 4.2.3.5) (Figure 16) (FLOSS et al. 1972; BORNEMANN et al. 1996, 2000; JAKEMAN et al. 1998; LEWIS et al. 1999; OSBORNE et al. 2000; IUBMB 2001).

The biologically unique conversion of choris-mic acid to prephenic acid by chorismate mutase (EC 5.4.99.5) is formally a Claisen rearrangement reaction (Figures 17 and 18). Decarboxylation and loss of hydroxyl group from prephenic acid (prephenate dehydratase, EC 4.2.1.51) yields phe-nylpyruvic acid, and the PLP-dependent transami-nation catalysed by aromatic aminotransferase

COOH CH2

CH2OP

HO H H OH H OH O

3-deoxy-7-phospho-D-arabino-heptulosonic acid

O O P

O OH HOOC

OH O

OH OH H

OP

OH

HOOC O

OH

OH HOOC

O

O OH

OH HOOC

O CH2

O OH

O HOOC

CH2

H OH H

O OH HOOC

CH2

H OH

O O

OH HOOC

H2C

H OH O

HO

OH COOH

OH O

NAD NADH + H

P

NAD NADH + H

3-dehydroquinic acid

O P

H B

H

3-dehydroquinic acid

HO

OH COOH

OH O

H2O

enzyme

B H

enzyme

N H

OH COOH

OH HO

N OH

OH COOH

enzyme

OH OH COOH

O

H2O

HO

OH COOH

OH N

H

H B

H₂O

3-dehydroshikimic acid enzyme NH2

Figure 13

OH OH COOH

PO

H2C COOH

OP

H

O OH COOH

PO

CH2

H

COOH O P

B

O OH COOH

PO

CH2

COOH

3-phosphoshikimic acid

phosphoenolpyruvic acid

5-(1-carboxyvinyl)-3-phosphoshikimic acid

P

COOH

PO OH

O COOH

CH2

COOH

OH O

COOH CH2

FMNH2 FMNH + P FMNH + P FMNH2+ H

5-(1-carboxyvinyl)-3-phosphoshikimic acid chorismic acid

COOH

OH O

COOH CH2

(EC 2.6.1.57) forms phenylalanine. In the presence of a NAD+_{-dependent dehydrogenase} (prephen-ate dehydrogenase, EC 1.3.1.12), or a NADP+ -dependent prephenate dehydrogenase (NADP+₎

(EC 1.3.1.13) decarboxylation occurs with reten-tion of the hydroxyl funcreten-tion. Transaminareten-tion of the resultant 4-hydroxyphenylpyruvic acid by aromatic-amino-acid transaminase (EC 2.6.1.57)

Figure 16 Figure 15

Figure 17

EC 1.3.1.13 EC 1.3.1.43

COOH O

phenylpyruvic acid

COOH

NH2

L-phenylalanine

COOH NH2

HO COOH

O HO

chorismic acid prephenic acid L-arogenic acid

O COOH

OH

COOH CH2

COOH O

O

H

O

OH

HOOC

OH

COOH NH2

4-hydroxyphenylpyruvic acid L-tyrosine

EC 5.4.99.5

EC 1.3.1.12

EC 4.2.1.51 EC 4.2.1.91

EC 2.6.1.57

EC 2.6.1.57 EC 2.6.1.57

H2O + CO2

H H

NAD

NADH + H + CO2

NAD(P)

NAD(P)H + H + CO2

CO2

H2O +

L-glutamic acid 2-oxoglutaric acid

L-glutamic acid 2-oxoglutaric acid

L-glutamic acid 2-oxoglutaric acid

54

OH HOOC

O COOH

pseudo-diequatorial pseudo-diaxial

prephenic acid chorismic acid conformers

OH

COOH O

HOOC

O O

H HOOC CH2 _COOH COOH

O H

O CH2

O O H

or tyrosine transaminase (EC 2.6.1.5) subsequently gives tyrosine (CLARK et al. 1990; IUBMB 2001; MARTÍ et al. 2003).

L-Arogenic acid is the result of transamination of prephenic acid occurring prior to the decar-boxylation, and can be transformed into both phenylalanine and tyrosine depending on the absence or presence of a suitable enzyme activity, e.g. activity of arogenate dehydratase (EC 4.2.1.91) and arogenate dehydrogenase (EC 1.3.1.43), re-spectively (IUBMB 2003). In animals, which lack this pathway, direct hydroxylation of phe-nylalanine to tyrosine (and of tyrosine to DOPA) may be achieved. This reaction is catalysed by (6R)-5,6,7,8-tetrahydro-L-biopterin (L-erythro -5,6,7,8-tetrahydrobiopterin, BH₄)-dependent hy-droxylase enzyme (phenylalanine hyhy-droxylase, EC 1.14.16.1)4_{, the hydroxyl oxygen being derived} from molecular oxygen. The second oxygen atom

is reduced to water (Figure 19). The rationalised reaction mechanism is given in Figure 20 (CARR et al. 1995; IUBMB 2001).

3.2 Tryptophan

Tryptophan is derived from chorismic acid via anthranilic (2-aminobenzoic) acid in a reaction catalysed by anthranilate synthase (EC 4.1.3.27) (Figure 21). In some organisms this enzyme is a part of a multifunctional protein having more other enzymatic activities for the biosynthesis of tryp-tophan, i.e. anthranilate phosphoribosyltransferase (EC 2.4.2.18), phosphoribosylanthranilate isomer-ase (EC 5.3.1.24), indole-3-glycerol-phosphate synthase (EC 4.1.1.48), and tryptophan synthase (EC 4.2.1.20) activity (IUBMB 2001).

Chorismic acid is first aminated at C-2 by L-glu- tamine to give 2-amino-2-deoxyisochorismic acid,

HO COOH

NH2

COOH

NH₂

L-phenylalanine L-tyrosine

H NH2 COOH

O _H

H

COOH

NH2

O

H H

H

4_{The product of tetrahydrobiopterin reduction is 4a-hydroxytetrahydrobiopterin. It can dehydrate 6,7-dihydrobiopterin,}

both spontaneously and by the action of 4a-hydroxytetrahydropterin dehydratase (EC 4.2.1.96). The 6,7-dihydrobiopterin can be enzymatically reduced back to tetrahydrobiopterin by 6,7-dihydropteridine reductase (EC 1.5.1.34), or spontaneously rearranges to the more stable 7,8-dihydrobiopterin.

Figure 18

Figure 19

Figure 20

5,6,7,8-tetrahydrobiopterin 7,8-dihydrobiopterin

N N

N

N NH2

O H₃C

OH OH

H 1 2

3 4 5 6 7 8

4a

8a N N

NH

N NH2

O H₃C

OH

OH _H

H

HO

COOH

NH2

COOH

NH₂

L-phenylalanine

EC 1.14.16.1

L-tyrosine

+ +

which yields anthranilic acid by elimination of the C-4 hydroxyl as water and C-3 substituent as pyruvic acid (Figure 22). The transformation in-volves an S_N2'-type of reaction, incoming ammonia, generated from glutamine, acts as a nucleophile attacking the diene system.

5-Phospho-α-D-ribose 1-diphosphate is the ac-tivated form of ribose. It forms from either AMP in a reaction catalysed by adeninephosphoribosyl transferase (EC 2.4.2.7) or from D-ribose 5-phos-phate (forming in pentose phos5-phos-phate pathway) and ATP by ribose-phosphate diphosphokinase

pyruvic acid

N

COOH

NH2

H

O

OH OH

O P P

CH2

O

P

COOH NH2

P P

5-phospho-�-D-ribose 1-diphosphate

EC 2.4.2.18

O

OH OH CH2

O

P N

HOOC

H

L-tryptophan (1S,2R)-1-(indol-3-yl)glycerol 3-phosphate 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate COOH

OH

O COOH

CH2

Gln Glu

EC 4.1.3.27

N-(5-phospho-�-D-ribosyl)anthranilic acid

chorismic acid anthranilic acid

EC 5.3.1.24

H₂O+CO₂

EC 4.1.1.48

D-glyceraldehyde 3-phosphate

EC 4.2.1.20 Ser

H3C COOH

O

CH O

CH2OP

H OH

COOH

NH HO

O OH

OP

N H

OP

OH

OH

Figure 21

Figure 22

o

OH OH

PO

OPP

O

OH OH

PO

OH

AMP

adenine

D-ribose 5-phosphate

5-phospho-�-D-ribose 1-diphosphate

ATP AMP

EC 2.4.2.7 EC 2.7.6.1

PP

Figure 23

pyruvic acid

H3C COOH

O H2C COOH

OH

HOOC COOH

NH2

COOH NH2

O

H2N COOH

NH2

COOH

OH

O COOH CH2

chorismic acid 2-amino-2-deoxyisochorismic acid anthranilic acid

COOH

NH2

CH2

COOH O

56

(EC 2.7.6.1) (Figure 23). It has also a key posi-tion in the biosynthesis of histidine and purine nucleotides (IUBMB 2003).

The tryptophan synthase multienzyme complex (EC 4.2.1.20) catalyses the final step of tryp-tophan biosynthesis in its two subunits. In the first enzyme subunit, (1S,2R)-1-(indol-3-yl)glycerol 3-phosphate splits off D-glyceraldehyde 3-phos-phate and yields indole. Indole moves to the sec-ond enzyme subunit where it reacts with the dehydrated Schiff base of L-serine yielding the tryptophan-PLP Schiff base, which is then hy-drolysed to the parent compounds, tryptophan and PLP (Figure 24).

3.3 Histidine

The biosynthetic pathway of L-histidine is unu-sual in that five of its carbon atoms are produced from 5-phospho-α-D-ribose 1-diphosphate, the key intermediate of tryptophan biosynthesis. One carbon atom comes from ATP (Figure 25).

The early stages of histidine biosynthesis involve condensation of 5-phospho-α_-D-ribose 1-di-phosphate with ATP, followed by elimination of diphosphoric acid, which is catalysed by ATP phosphoribosyltransferase (EC 2.4.2.17). Elimi-nation of diphosphoric acid from this product, N1_{-5'-phosphoribosyl-ATP, is catalysed by} phos-phoribosyl-ATP diphosphatase (EC 3.6.1.31) and yields N'-5'-phosphoribosyl-AMP. The pyrimidine ring of this compound opens by phosphoribo-syl-AMP cyclohydrolase (EC 3.5.4.19) under the formation of N'-5'-phosphoribosylformimino-5-ami- no-imidazole-4-carboxamide ribonucleotide. Then 1-(5-phosphoribosyl)-5-[_{(phosphoribosyl} amino)methylene amino] imidazole-4-carboxa-mide isomerase (EC 5.3.1.16) splits the ribose attached at N1 _{to form 5-amino-4-carboxamide} ribonucleotide and the intermediate fragment, which reacts with ammonia from glutamine to form 3-(imidazol-4-yl)-1-glycerol phosphate. The reaction catalysed by imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) converts this compound

HO COOH

NH2

indole

L-tryptophan

(1S,2R)-1-(indol-3-yl)glycerol 3-phosphate

N H

OP

OH

OH H

CH

CH2OP

H OH

O

N

OP

O

OH H

H

N H

N HO

H3C

OP

N COOH H2C

H D-glyceraldehyde 3-phosphate

N H

N HO

H3C

OP

COOH

N

H H

H

NH

COOH

N

OP

H3C HO

N

H2O

N

COOH

NH2 H

H2O

N HO

H3C

OP

H O

H2O

N HO

H3C

OP

N COOH HO

pyridoxal 5´-phosphate Schiff base Schiff base

L-serine

to 3-(imidazol-4-yl)-2-oxopropyl phosphate, which yields, in a transamination reaction catalysed by a PLP protein histidinol-phosphate transaminase (EC 2.6.1.9), L-histidinol phosphate. Histidinol phosphate is hydrolysed to L-histidinol by his-tidinol-phosphatase (EC 3.1.3.15) and histidinol is finally oxidised to L-histidine by the NAD-de-pendent histidinol dehydrogenase (EC 1.1.1.23) (IUBMB 2001).

CH2

OH OH

O

PO

PO

OH OH

O

N N N N

NH

H CH2

OH OH

O

PO

N N N NH2

O N O OH OH

PO H

CH2

OH OH

O

PO

N N N NH2

O N OH

PO OH O NH

H

OPP PO

OH OH

O

CH2

OH OH

O

PPPO N

N N N

CH2

OH OH

O

PPPO

PO

OH OH

O

N N N N

NH

EC 2.4.2.17

PP _{H2O PP}

EC 3.6.1.31

H2O

EC 3.5.4.19

EC 5.3.1.16

CH2

OH OH

O

PO

N N NH2

O H2N

H N

OH

PO OH O

H O

Gln

Glu

ATP

5-phospho-�-D-ribose

1-diphosphate

N

OH OP

NH N NH

OP

O

NH

N OP

NH2

NH

N OH

NH2

NH N

COOH NH2

NH

N OP

OH OH

H

EC 4.2.1.19

3-(imidazol-1-yl)-2-oxopropylphosphate

L-glutamic acid

2-oxoglutaric acid

L-histidinol phosphate L-histidinol

L-histidine

EC 2.6.1.9

EC 3.1.3.15 EC 1.1.1.23

H2O

P

2 NAD 2 NADH + H

EC (Enzyme Commission) numbers and some common abbreviations

EC (Enzyme Commission) numbers, assigned by IUPAC-IUBMB, were taken from KEGG: Kyoto Ency-clopedia of Genes and Genomes, http://www.biologie. uni-hamburg.de. In many structures, the abbreviation P is used to represent the phosphate group and PP the diphosphate group. At physiological pH, these

58

and some other groups will be ionized, but in pictures the unionised forms are depicted to simplify the structures, to eliminate the need for counter-ions, and to avoid the mechanistic confusion.

ADP – denosine 5'-diphosphate AMP – adenosine 5'-monophosphate APS – adenosine-5'-phosphosulfuric acid ATP – adenosine 5'-triphosphate

CoA – coenzyme A as a part of a thioester FMN – flavin mononucleotide

GSH – glutathione (reduced) GSSG – glutathione (oxidised)

NADH – nicotinamide adenine dinucleotide NADPH – nicotinamide adenine dinucleotide phos-

phate

P – phosphoric acid

PAPS – 3'-phosphoadenosine-5'-phosphosulfate PLP – pyridoxal 5'-phosphate

PP – diphosphoric acid

References

BENDER S.L., MEHDI S., KNOWLES J.R. (1989): Dihyd-roquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis. Biochemistry, 28: 7555–7560.

BORNEMANN S., LOWE D., THORNELEY R.N. (1996): The transient kinetics ofEscherichia coli chorismate synthase: substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate. Biochemistry, 35: 9907–9916.

BORNEMANN S., THEOCLITOU M.E., BRUNE M., WEBB M.R., THORNELEY R.N., ABELL C.A. (2000): A secondary

β _{deuterium kinetic isotope effect in the chorismate}

syn-thase reaction. Bioorganic Chemistry, 28: 191–204. CARR R.T., BALASUBRAMANIAN S., HAWKINS P.C.,

BEN-KOVIC S.J. (1995): Mechanism of metal independent hydroxylation by Chromobacterium violaceum phenyl-alanine hydroxylase. Biochemistry, 34: 7525–7532. CLARK T., STEWART J.D., GANEM B. (1990):

Transiti-on-state analogue inhibitors of chorismate mutase. Tetrahedron, 46: 731–748.

FLOSS H.G., ONDERKA D.K., CARROLL M. (1972): Stereo-

chemistry of the 3-deoxy-D-arabino-heptulosonate

7-phosphate synthetase reaction and the chorismate synthetase reaction. Journal of Biological Chemistry,

247: 736–744.

GOURLEY D.G., SHRIVE A.K., POLIKARPOV I., KRELL T., COGGINS J.R., HAWKINS A.R., ISAACS N.W., SAWYER L. (1999): The two types of 3-dehydroquinase have dis-tinct structures but catalyze the same overall reaction. Nature Structural Biology, 6: 521–525.

IUBMB (2001): http://www.chem.qmul.ac.uk/iubmb/en-zyme/reaction/.

IUBMB (2002): http://www.chem.qmul.ac.uk/iubmb/en-zyme/reaction/.

IUBMB (2003): http://www.chem.qmul.ac.uk/iubmb/en-zyme/reaction/.

JAKEMAN D.L., MITCHELL D.J., SHUTTLEWORTH W.A., EVANS J.N. (1998): On the mechanism of 5-enolpyru-vylshikimate-3-phosphate synthase. Biochemistry, 37: 12012–12019.

JOST R., BERKOWITZ O., WIRTZ M., HOPKINS L., HAWKES-FORD M.J., HELL R. (2000): Genomic and functio-nal characterization of the oas gene family encoding

O-acetylserine(thiol)lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana. Gene, 253: 237–247.

LEWIS J., JOHNSON K.A., ANDERSON K.S. (1999): The catalytic mechanism of EPSP synthase revised. Bio-chemistry, 38: 7372–7379.

MARTÍ S., ANDRÉS J., MOLINER V., SILLA E., TUŇÓN I., BERTRÁN J. (2003): Conformational equilibrium of chorismate. A QM/MM theoretical study combining statistical simulations and geometry optimistations in gas phase and in aqueous solution. Journal of Molecular

Structure (Theochem),632: 197–206.

OSBORNE A., THORNELEY R.N., ABELL C., BORNEMANN S. (2000): Studies with substrate and cofactor analo-gues provide evidence for a radical mechanism in the chorismate synthase reaction. Journal of Biological Chemistry, 275: 35825–35830.

SENGBUSCH P.: Botany Online – The Internet Hypertext-

book. http://biologie.uni-hamburg.de/b_online/e00/

default.htm.

Received for publication June 13, 2005 Accepted after corrections September 26, 2005

Corresponding author:

Prof. Ing. JAN VELÍŠEK, DrSc., Vysoká škola chemicko-technologická v Praze, Fakulta potravinářské a biochemické technologie, Ústav chemie a analýzy potravin, Technická 5, 166 28 Praha 6, Česká republika