Czech J. Food Sci. Vol. 24, No. 1: 1–10

Biosynthesis of Food Constituents: Amino Acids: 1. The Glutamic Acid and Aspartic Acid 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: 1. The glutamic acid and aspartic acid groups – a review. Czech J. Food Sci., 24: 1–10.

This review article gives a survey of principal pathways that lead to the biosynthesis of the proteinogenic amino acids of the glutamic acid group (glutamic acid, glutamine, proline, arginine) and aspartic acid group (aspartic acid, aspar- agine, threonine, methionine, lysine, isoleucine) starting with oxaloacetic acid from the citric acid cycle. There is an extensive use of reaction schemes, sequences, and mechanisms with the enzymes involved and detailed explanations using sound chemical principles and mechanisms.

Keywords: biosynthesis; amino acids; glutamic acid; glutamine; proline; arginine; aspartic acid; asparagine; threonine; methionine; lysine; isoleucine

Commonly, 20 L-amino acids encoded by DNA aspartic acid, cysteine, glutamic acid, glutamine, represent the building blocks of animal, plant, glycine, proline, serine, tyrosine). Arginine and and microbial proteins, and a limited number histidine are classified as essential, sometimes of amino acids participate in the biosynthesis of as semi-essential amino acids, as their amounts certain shikimate metabolites and particularly in synthesised in the body is not sufficient for normal the formation of alkaloids. The basic amino acids growth of children. Although it is itself non-essen- encountered in proteins are called proteinogenic tial, cysteine (sometimes classified as conditionally amino acids. The biosynthesis of some of these essential amino acid) can partly replace methio- amino acids proceeds by ribosomal processes only nione which is an essential amino acid. Similarly, in microorganisms and plants while the ability tyrosine can partly replace phenylalanine. to synthesise them is lacking in animals includ- ing human beings. These amino acids have to be 1 THE GLUTAMIC ACID GROUP obtained in the diet (or produced by hydrolysis of body proteins) since they are required for normal 1.1 Glutamic acid and glutamine good health; they are referred to as essential amino acids. The essential amino acids are arginine, Free ammonium ions are toxic to living cells and histidine, isoleucine, leucine, lysine, methionine, are rapidly incorporated into organic compounds. phenylalanine, threonine, tryptophan, and valine. One of such transformations is the reaction of The rest of the encoded amino acids are referred to ammonia with 2-oxoglutaric acid from the citric as non-essential amino acids (alanine, asparagine, acid cycle to produce L-glutamic acid. This reac-

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

1 Vol. 24, No. 1: 1–10 Czech J. Food Sci.

Citric acid cycle 2-oxoglutaric acid

L-glutamic acid L-ornithine L-arginine

L-glutamine L-proline L-citrulline

Figure 1 tion is known as reductive amination. Glutamic catalysed by glutamine synthetase (EC 6.3.1.2) and acid is, accordingly, the first amino acid generated. proceeds through the intermediate L-glutam-5-yl Glutamic acid itself can bind a further ammonium phosphate-ATP which splits off ADP and phos- ion to form L-glutamine, the second amino acid. phoric acid (Figure 3). Glutamic acid then serves as the precursor of Very important for many amino acid syntheses L-proline, L-ornithine, L-citrulline, and L-argi- is the group of enzymes called transaminases. nine. The biosynthetic pathway of the glutamic They are able to transfer an amino group (mostly acid amino acid family is schematically shown in that of glutamine) to a 2-oxo acid according to the Figure 1 (SENGBUSCH). following equation: The formation of glutamic acid (as well as the reverse rection) is catalysed by glutamate dehy- (amino acid)1 + (2-oxo acid)2 = (2-oxo acid)1+ drogenase. Nicotinamide adenine dinucleotide + (amino acid)2 (NADH)-dependent glutamate dehydrogenase (EC Thus, the glutamine amino group is transferred 1.4.1.2) or the enzyme using nicotinamide adenine to 2-oxoglutaric acid (Figure 4) producing glutamic dinucleotide phosphate (NADPH) as the coenzyme acid. The reaction is catalysed by glutamate syn- (EC 1.4.1.4) is employed as hydrogen donor by thases having either NADPH (EC 1.4.1.13) or microorganisms (e.g. Saccharomyces cerevisiae). NADH (EC 1.4.1.14) as cofactor. 1 In plants is the reaction mainly dependent on the The transamination reaction catalysed by trans- coenzyme NAD(P)H (EC 1.4.1.3) (Figure 2). aminases is dependent on the coenzyme pyridoxal L-Glutamine arises as a product of amidation of 5'-phosphate (PLP). The reaction starts with the glutamic acid that binds a further ammonium ion addition of the unprotonised amino group of an (SENGBUSCH). This amidation of glutamic acid is amino acid to the electron deficient carbon of

NAD(P)H + H NADP

HOOC COOH HOOC COOH + H O + NH3 2 O EC 1.4.1.3 NH2

2-oxoglutaric acid L-glutamic acid

Figure 2 ADP ATP NH3 P O O HOOC COOH COOH PO COOH H2N NH2 EC 6.3.1.2 NH2 EC 6.3.1.2 NH2

L-glutamic acid L-glutam-5-yl phosphate L-glutamine

Figure 3

1Plants take up nitrogen as nitrates and in smaller amounts also as ammonium ions. A few species, mainly leguminose plants, live in symbiosis with nitrogen-fixing bacteria that are able to reduce atmospheric nitrogen. Microorganisms mainly require ammonium ions as the nitrogen source, some bacteria and moulds are able to use nitrates similarly to plants. Plants reduce nitrates in two steps. In the first step, nitrate is reduced to nitrite by nitrate reductase (NADH) (EC 1.7.1.1). In the second step, nitrite is reduced to ammonia by ferredoxin-nitrite reductase (EC 1.7.7.1).

2 Czech J. Food Sci. Vol. 24, No. 1: 1–10

NADPH + H O NADP COOH HOOC COOH HOOC COOH HOOC COOH H N 2 + + NH2 O NH NH EC 1.4.1.13 2 2

L-glutamine 2-oxoglutaric acid L-glutamic acid L-glutamic acid

Figure 4 the polarised carbonyl group of PLP under the 5-semialdehyde formed yields spontaneously a formation of a monotopic carbinolamine. The cyclic Schiff base (S)-1-pyrroline-5-carboxylic carbinolamine dehydration then leads to an aldi- acid, i.e. (S)-3,4-dihydro-2H-pyrrole-2-carboxylic mine (Schiff base). The α-hydrogen atom of the acid. Its reduction to proline is achieved by pyr- original amino acid in the aldimine is now much roline-5-carboxylate reductase (EC 1.5.1.2) and more acidic and can be readily eliminated. Isom- the hydrogen needed is supplied by NAD(P)H. erisation of the resulting intermediate leads to a An alternative pathway of proline biosynthesis is ketimine (an imine of PLP with an 2-oxo acid). a cyclisation of L-ornithine catalysed by ornithine Hydrolysis of this ketimine generates pyridoxamine cyclodeamidase (EC 4.3.1.12) (Figure 6). 5'-phosphate and the 2-oxo acid (Figure 5). The amino group of pyridoxamine 5'-phosphate is then 1.3 Arginine transferred to another 2-oxo acid molecule and PLP is regenerated (DEWICK 2002). The biosynthesis of the essential amino acid L-arginine in microorganisms and plants starts 1.2 Proline from L-glutamic acid (IUBMB 2001). It proceeds via L-ornithine in several intermediate steps and L-Proline is generated from L-glutamic acid by needs the reduction of the γ-carboxyl group to a ring formation (IUBMB 2001). The reaction is carbonyl group under the participation of ATP facilitated by a preliminary phosphorylation of similarly to proline biosynthesis. To prevent the glutamic acid by γ-glutamyl kinase (EC 2.7.2.11) spontaneous cyclisation of the semialdehyde, the that leads to an unstable intermediate L-glutam-5-yl first step is acetylation of glutamic acid with acetyl- phosphate under consumption of one molecule CoA catalysed by amino-acid N-acetyltransferase of ATP. The reduction of the phosphorylated (EC 2.3.1.1). The carboxyl group of the intermediate γ-carboxyl to a carbonyl group is catalysed by N-acetyl-L-glutam-5-yl phosphate thus formed is the NAD(P)H dependent glutamate semialde- then reduced to a carbonyl group by NADPH-de- hyde dehydrogenase (EC 1.2.1.41). Glutamic acid pendent N-acetyl-γ-glutamyl-phosphate reductase

R COOH R COOH H H O HO NH N

OH OH OH RCOOH PO PO PO + H2O H + NH2 N CH3 N CH3 N CH3 H 2-amino acid pyridoxal 5´-phosphate vicinal carbinolamine aldimine

R COOH R COOH

NH2 N H N

OH OH OH RCOOH PO PO PO + O N CH N CH 3 3 N CH3 H 2-oxo acid pyridoxamine 5´-phosphate ketimine

Figure 5

3 Vol. 24, No. 1: 1–10 Czech J. Food Sci.

ATP ADP NAD(P)H NAD(P) + P O H HOOC COOH PO COOH O COOH NH 2 EC 2.7.2.11 NH2 EC 1.2.1.41 NH2

L-glutamic acid L-glutam-5-yl phosphate L-glutamic acid 5-semialdehyde

NH3 2 NAD(P) 2 NAD(P)H H2O

COOH H2N H H N COOH NH2 H N COOH EC 1.5.1.2 EC 4.3.1.12 H

L-ornithine L-proline (S)-1-pyrroline-5-carboxylic acid

Figure 6

(EC 1.2.1.38) and the next intermediate N-acetyl- L-argininosuccinic acid. Argininosuccinate lyase L-glutamic acid 5-semialdehyde is transformed to (EC 4.3.2.1) then splits this intermediate to arginine N-acetyl-L-ornithine in a transamination reaction and fumaric acid (Figure 8). The release of urea catalysed by a PLP protein N2-acetylornithine from arginine is catalysed by arginase (EC 3.5.3.1) 5-transaminase (EC 2.6.1.11). Finally, hydrolysis and generates ornithine. of N-acetyl-L-ornithine to L-ornithine is cata- lysed by acetylornithine deacetylase (EC 3.5.1.16) 2 THE ASPARTIC ACID GROUP (Figure 7). Ornithine functions as an acceptor of carbamoyl 2.1 Aspartic acid and asparagine phosphate in the urea (ornithine) cycle. At the same time, a phosphoric acid is split off and the resulting L-Aspartic acid is generated by transamination of next intermediate is L-citrulline. Both intermediates oxaloacetic acid (oxosuccinic acid) from the citric belong to the group of amino acids that is common acid cycle. From aspartic acid branches a pathway in cells but is not used in the synthesis of proteins. to L-homoserine, an intermediate that is the start- The reaction is catalysed by ornithine carbamoyl ing point of the biosyntheses of L-threonine and transferase (EC 2.1.3.3). The next reaction leading L-methionine. The biosynthesis of methionine from citrulline to L-argininosuccinic acid is catalysed requires the sulphur transfer from L-cysteine. by argininosuccinate synthase (EC 6.3.4.5). Oxygen From aspartic acid then lead pathways to L-lysine of the ureido group of citrulline is activated with and the branched amino acid L-isoleucine. Biosyn- ATP via the intermediate citrullyl-AMP; AMP is theses of both these amino acid require pyruvic replaced by amino group of aspartic acid forming acid from the glycolytic pathway (AZEVEDO et al.

acetyl-CoA CoA ATP ADP O HOOC COOH HOOC COOH COOH PO

NH H C NH H3C NH 2 EC 2.3.1.1 3 EC 2.7.2.8 O O L-glutamic acid N-acetyl-L-glutamic acid N-acetyl-L-glutam-5-yl phosphate

NADP + P

EC 1.2.1.38

2-oxoglutaric acid L-glutamic acid NADPH + H H3CCOOH H2O H COOH COOH COOH H2N H2N O NH H C NH H C NH 2 3 EC2.6.1.11 3 EC 3.5.1.16 O O

L-ornithine N-acetyl-L-ornithine N-acetyl-L-glutamic acid 5-semialdehyde

Figure 7

4 Czech J. Food Sci. Vol. 24, No. 1: 1–10

O

H2N P carbamoyl phosphate ATP PP P O AMP COOH COOH O H2N H2N N COOH H NH NH HN N 2 EC 2.1.3.3 2 EC 6.3.4.5 H NH2 L-ornithine L-citrulline NH2 O L-citrullyl-AMP HOOC COOH H2N NH2 L-aspartic acid urea EC 3.5.3.1 EC 6.3.4.5 COOH H O HOOC 2 COOH fumaric acid COOH AMP NH H N COOH COOH H2N N HN N H H NH NH 2 EC 4.3.2.1 2 L-arginine L-argininosuccinic acid

Figure 8

1997; SENGBUSCH). The biosynthetic pathway of inase (EC 2.6.1.1), an enzyme dependent on the the aspartic acid amino acid family is schemati- coenzyme PLP (Figure 10). cally shown in Figure 9. L-Asparagine forms from aspartic acid by the The transamination of oxaloacetic acid to L-aspar- addition of a further amino group. This may be tic acid is a reaction catalysed by aspartate transam- achieved by the fixation of an ammonium ion

Citric acid cycle oxaloacetic acid

L-asparagine L-aspartic acid

L,L-2,6-diaminopimelic acid

L,L-cystathionine L-homoserine pyruvic acid

L-homocysteine L-lysine

Glycolysis L-threonine

L-methionine

pyruvic acid

L-isoleucine

Figure 9

O HOOC COOH COOH HOOC COOH HOOC HOOC + + NH COOH 2 EC 2.6.1.1 NH2 O

oxaloacetic acid L-glutamic acid L-aspartic acid 2-oxoglutaric acid

Figure 10

5 Vol. 24, No. 1: 1–10 Czech J. Food Sci.

ATP AMP + PP O HOOC COOH COOH COOH H2N COOH HOOC H N + 2 + NH NH2 NH2 EC 6.3.5.4 O NH2 2

L-aspartic acid L-glutamine L-asparagine L-glutamic acid

Figure 11

(analogous to the synthesis of glutamine). This dehydrogenase (EC 1.1.1.3) catalyses the conver- reaction (in some microorganisms) is catalysed sion of L-aspartic acid 4-semialdehyde to L-ho- by ammonia-dependent asparagine synthetas- moserine in the presence of coenzyme NADH es, e.g. asparagine synthetase (AMP forming) or NADPH (in plants). Homoserine kinase (EC (EC 6.3.1.1) or asparagine synthetase (ADP form- 2.7.1.39) catalyses the reaction common to threo- ing) (EC 6.3.1.4). On the other hand, asparagine nine, isoleucine, and methionine synthesis, in which may also form by the transfer of one amino group homoserine is converted in the presence of ATP to from glutamine to aspartic acid. In plants and ani- yield O-phospho-L-homoserine (4-phospho-L-ho- mals, however, all the asparagine synthases so far moserine) and ADP. The last step involves the characterised belong to the glutamine-dependent irreversible hydrolysis of O-phosphohomoserine class (GÁLVEZ-VALDIVIESO 2005). This amidation into threonine, which is catalysed by the enzyme is catalysed by asparagine synthetase (EC 6.3.5.4) threonine synthase (EC 4.2.3.1) (AZEVEDO et al. and the required energy supplied by ATP that yields 1997; IUBMB 2001). AMP and diphosphoric acid (Figure 11). 2.3 Methionine 2.2 Threonine The biosynthesis of the essential amino acid L-me- The biosynthesis of the essential amino acid thionine starts with L-homoserine and involves the threonine in microorganisms and plants consists transfer of sulphur from the C3 skeleton of L-cysteine of five steps and consumes two molecules of ATP, to the C4 skeleton of L-homoserine (Figure 13). one NAD(P)H + H+, and one NADH + H+ (Fig- This process termed trans-sulphuration is closely ure 12). Aspartate kinase (EC 2.7.2.4) is the first related to the biosynthesis of cysteine from ser- enzyme of the pathway catalysing the conversion ine and sulphide. Plants from most of the major of aspartic acid into L-aspart-4-yl phosphate (β-as- phylogenetic divisions were only observed to uti- partyl phosphate). L-Aspart-4-yl phosphate is lise O-phospho-L-homoserine (some bacteria use reduced to L-aspartic acid 4-semialdehyde (β-as- O-succinyl-L-homoserine, yeasts and fungi utilise partic acid semialdehyde) by aspartate-semialde- O-acetyl-L-homoserine). (AZEVEDO et al. 1997; hyde dehydrogenase (EC 1.2.1.11). Homoserine HESSE & HOEFGEN 2003).

ATP ADP NADPH + H NADP + P

COOH PO COOH O COOH HOOC O NH NH2 EC 2.7.2.4 2 EC 1.2.1.11 H NH2

L-aspartic acid L-aspart-4-yl phosphate L-aspartic acid 4-semialdehyde

NAD(P)H

EC 1.1.1.3

NAD(P) + H P ADP ATP OH H2O HO COOH COOH PO COOH H3C NH2 NH2 NH2 EC 4.2.3.1 EC 2.7.1.39

L-threonine O-phospho-L-homoserine L-homoserine

Figure 12

6 Czech J. Food Sci. Vol. 24, No. 1: 1–10

ADP PO COOH L-cysteine

NH2 ATP P O-phospho-L-homoserine EC 2.5.1.48 EC 2.7.1.39 NH2 HO COOH S COOH HOOC

NH2 NH2

L-homoserine EC 2.3.1.46 EC 2.5.1.48 L,L-cystathionine H2O

EC 4.4.1.8 succinyl-CoA HOOC O COOH HOOC COOH H3C COOH HS-CoA O NH2 L-cysteine succinic acid + NH O 3 O-succinyl-L-homoserine pyruvic acid

H4PteGlu 5-methyl- H4PteGlu COOH S COOH HS COOH HOOC H3C

NH2 NH2 EC 2.1.1.13 NH2

L-aspartic acid L-methionine L-homocysteine

Figure 13

Homoserine is transformed to O-phosphohomo- biosynthesis in plants whereas bacteria use the serine in a reaction catalysed by homoserine kinase monoglutamic acid form. The function of this (EC 2.7.1.39) which serves as the starting compound enzyme is both de novo synthesis of methionine for methionine biogenesis in plants. In microor- and the regeneration of S-adenosyl-L-methionine ganisms, the formation of O-succinylhomoserine (SAM) from S-adenosyl-L-homocysteine (SAH) is catalysed by homoserine O-succinyltransferase after methylation reactions. (EC 2.3.1.46). Cystathionine γ-lyase (EC 2.5.1.48), Eventually, about 20% of methionine in plants is the first enzyme unique to methionine biosynthe- incorporated into proteins and 80% is converted sis, catalyses the synthesis of L,L-cystathionine to SAM, the end product of the methionine bio- from cysteine and either O-phosphohomoserine synthetic pathway. The biosynthesis of SAM from or O-succinylhomoserine. Cystathionine-β-lyase methionine and ATP is catalysed by methionine (EC 4.4.1.8) catalyses the β-cleavage of cystath- adenosyltransferase (EC 2.5.1.6) (DEWICK 2002) ionine to yield homocysteine, pyruvic acid and (Figure 14). ammonia. The final reaction of methionine bio- synthesis, the methylation of homocysteine, is 2.4 Lysine catalysed by homocysteine methyltransferase (EC 2.1.1.13). The methyl group is derived from The enzyme dihydrodipicolinate synthase N5-methyltetrahydropteroylglutamic acid (5-me- (EC 4.2.1.52) is the first enzyme of the essential thyl-H4PteGlu) in a cobalamin dependent reaction. amino acid L-lysine biosynthesis branch of the It appears that the triglutamic acid form, 5-methyl- aspartic acid metabolic pathway (AZEVEDO et al.

H4PteGlu3, is the methyl donor for methionine 1997; BORN & BLANCHARD 1999; IUBMB 2001).

NH2 Dihydrodipicolinate synthase catalyses the al- dolisation of L-aspartic acid 4-semialdehyde and ATP + H + H2O PP + P N N H3C H3C pyruvic acid from the glycolytic pathway. The S S CH2 O N N product of this aldolisation reaction spontaneously EC 2.5.1.6 forms an intermediate cyclic Schiff base under NH NH 2 2 OH OH the elimination of water. Its dehydration yields COOH COOH (R)-2,3-dihydropyridine-2,6-dicarboxylic acid

L-methionine S-adenosyl-L-methionine (2,3-dihydrodipicolinic acid). Dihydrodipicolinate reductase (EC 1.3.1.26) catalyses the NAD(P)H-de- Figure 14 pendent reduction of 2,3-dihydrodipicolinic acid

7 Vol. 24, No. 1: 1–10 Czech J. Food Sci. to (R)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylic thesis involves the PLP-dependent decarboxylation acid (2,3,4,5-tetrahydrodipicolinic acid). The tet- of meso-2,6-diaminopimelic acid to lysine, which rahydropyridine ring then opens and the released is catalysed by the enzyme diaminopimelate de- amino group is succinylated by 2,3,4,5-tetrahydro- carboxylase (EC 4.1.1.20) (Figure 15). pyridine-2,6-dicarboxylate N-succinyltransferase (EC 2.3.1.117) yielding (R)-2-succinylamino-6-oxo- 2.5 Isoleucine heptanedioic acid (N-succinyl-L-2-amino-6-oxo- pimelic acid). Transamination of this oxo acid, The first step of the essential amino acid L-iso- catalysed by the PLP-dependent succinyldiami- leucine biosynthesis, catalysed by threonine am- nopimelate transaminase (EC 2.6.1.17), forms monia-lyase (EC 4.3.1.19) yields 2-oxobutyric N-succinyl-L,L-2,6-diaminoheptanedioic acid (2-oxobutanoic) acid and ammonia (Figure 16) (N-succinyl-L,L-2,6-diaminopimelic acid). Suc- (IUBMB 2003). This transformation of threonine cinyl-diaminopimelate desuccinylase (EC 3.5.1.18) to 2-oxobutanoic acid probably involves the initial splits off succinic acid from N-succinyl-L,L-2,6-di- elimination of water, followed by isomerisation aminopimelic acid yielding L,L-2,6-diaminohep- and hydrolysis of the product with C = N bond tanedioic acid (L,L-2,6-diaminopimelic acid). Di- breakage (Figure 17). aminopimelate epimerase (EC 5.1.1.7) catalyses The next step, the formation of (S)-2-ethyl-2-hy- isomerisation of L,L-2,6-diaminopimelic acid to droxy-3-oxobutanoic acid (also known as 2-aceto- meso-2,6-diaminoheptanedioic acid (meso-2,6-di- 2-hydroxybutanoic acid), is catalysed by acetolac- aminopimelic acid). The last step in lysine biosyn- tate synthase (EC 2.2.1.6). This enzyme catalyses

H3C COOH

O pyruvic acid H2O OH

COOH O COOH HOOC COOH HOOC NH H NH NH 2 2 EC 4.2.1.52 O OH 2 HOOC N COOH

L-aspartic acid L-aspartic acid 4-semialdehyde

H2O HOOC COOH HS-CoA succinyl-CoA NAD(P) NAD(P)H + H

O NH O EC 1.3.1.26 EC 2.3.1.117 HOOC NCOOH HOOC NCOOH

HOOC 2,3,4,5-tetrahydrodipicolinic acid 2,3-dihydrodipicolinic acid N-succinyl-L-2-amino-6-oxopimelic acid

L-glutamic acid

EC 2.6.1.17

2-oxoglutaric acid HOOC COOH HOOC COOH H2O succinic acid

NH2 NH HOOC COOH O

EC 3.5.1.18 NH2 NH2 HOOC L,L-2,6-diaminopimelic acid

N-succinyl-L,L-2,6-diaminopimelic acid EC 5.1.1.7 CO2

H2N COOH HOOC COOH

NH NH NH 2 EC 4.1.1.20 2 2 L-lysine meso-2,6-diaminopimelic acid

Figure 15

8 Czech J. Food Sci. Vol. 24, No. 1: 1–10

H3C COOH

O NH3 pyruvic acid CO2 OH H3C COOH COOH H C H C H C 3 3 3 COOH NH EC 4.3.1.19 O EC 2.2.1.6 OH 2 O L-threonine 2-oxobutanoic acid (S)-2-ethyl-2-hydroxy-3-oxobutanoic acid

NADPH + H

EC 1.1.1.86

2-oxoglutaric acid L-glutamic acid NADP H2O

CH3 CH3 CH3

H3C COOH H3C COOH H3C COOH OH NH2 EC 2.6.1.42 O EC 4.2.1.9 OH

L-isoleucine (S)-3-methyl-2-oxopentanoic acid (2R,3R)-2,3-dihydroxy-3-methylpentanoic acid

Figure 16 the condensation of 2-oxobutanoic acid and pyruvic (methylhydroxyketol acid) in an alkyl migration acid and requires divalent metal ions, FAD, and (the alkyl migration is highly specific for Mg2+), thiamine diphosphate. Thiamine diphosphate me- which is followed by the NADPH-dependent re- diated decarboxylation of pyruvic acid produces duction in the presence of a divalent metal ion one acetaldehyde equivalent bound in the form (such as Mg2+, Mn2+ or Co2+) to yield (2R,3R)- of enamine which reacts as a nucleophile in the 2,3-dihydroxy-3-methylpentanoic acid (dihydroxy addition reaction with 2-oxobutanoic acid. The acid) (Figure 19). Dihydroxy-acid dehydratase subsequent release from the thiamine diphosphate (EC 4.2.1.9) catalyses the next step in isoleucine carrier generates (S)-2-ethyl-2-hydroxy-3-oxobu- biosynthesis which involves the dehydration and tanoic acid (Figure 18). tautomerisation of 2,3-dihydroxy-3-methylpen- Ketol-acid reductoisomerase (EC 1.1.1.86) ca- tanoic acid to (S)-3-methyl-2-oxopentanoic acid. talyses an unusual reaction in the biosynthesis of 3-Methyl-2-oxopentanoic acid is finally converted isoleucine in which (S)-2-ethyl-2-hydroxy-3-ox- to isoleucine in a transamination reaction catalysed obutanoic acid (acetohydroxy acid) is converted by branched-chain-amino-acid transaminase (EC to (R)-3-hydroxy-3-methyl-2-oxopentanoic acid 2.6.1.42) (AZEVEDO et al. 1997; LEE et al. 2005).

H2O H2O NH3 OH COOH COOH COOH COOH H C H3C 3 H3C H3C NH NH2 2 NH O

L-threonine 2-oxobutanoic acid

Figure 17

CH3 H OH O COOH H H H3C OH H3C H3C O 1 1 1 R R COOH R CH3 N S N S N S + H3C COOH OH 2 2 2 H3C R O H3C R H3C R thiamine diphosphate/pyruvic acid regenerated thiamine (S)-2-ethyl-2-hydroxy-3-oxobutanoic acid derived enamine diphosphate anion

Figure 18

9 Vol. 24, No. 1: 1–10 Czech J. Food Sci.

CH3 CH CH3 2 CH3 3 NADPH + H NADP CH 2 2 Mg CH CH2 M CH2 H CC CCOOH 3 H3CCCCOOH H3CCC COOH H3CC CH COOH O OH OO OH O OH OH H acetohydroxy acid transition state methylhydroxyketol acid dihydroxy acid

Figure 19

EC (Enzyme Commission) numbers and some rived amino acids in higher plants. Phytochemistry, common abbreviations 46: 395–419. BORN T.L., BLANCHARD J.S. (1999): Structure/function EC (Enzyme Commission) numbers, assigned by studies on enzymes in the diaminopimelate pathway IUPAC-IUBMB, were taken from KEGG: Kyoto Ency- of bacterial cell wall biosynthesis. Current Opinion in clopedia of Genes and Genomes, http://www.biologie. Chemical Biology, 3: 607–613. uni-hamburg.de. In many structures, the abbreviation DEWICK P.M. (2002): Medicinal natural products. A bio- P is used to represent the phosphate group and PP synthetic approach. 2nd Ed. Wiley, New York. the diphosphate group. At physiological pH, these GÁLVEZ-VALDIVIESO G., OSUNA D., MALDONADO J.M., and some other groups will be ionised, but in pictures PINEDA M., AQUILAR M. (2005): Purification of a func- the unionised forms are depicted to simplify the tional asparagines synthetase (PVAS2) from common structures, to eliminate the need for counter-ions, bean (Phaseolus vulgaris), a protein predominantly and to avoid the mechanistic confusion. found in root tissues. Plant Science, 168: 89–95. ADP adenosine 5'-diphosphate HESSE H., HOEFGEN R. (2003): Molecular aspects of AMP adenosine 5'-monophosphate methionine biosynthesis. Trends in Plant Science, 8: ATP adenosine 5'-triphosphate 259–262. CoA coenzyme A as part of a thioester LEE Y.T., TA H.T., DUGGLEBY R.G. (2005): Cyclopropane- FAD flavine adenine dinucleotide 1,1-dicarboxylate is a slow, tight-binding inhibitor of NADH nicotinamide adenine dinucleotide rice ketol-acid reductoisomerase. Plant Science, 168: NADPH nicotinamide adenine dinucleotide phos- 1035–1040. phate IUBMB (2001): http://www.chem.qmul.ac.uk/iubmb/en- P phosphoric acid zyme/reaction/. PLP pyridoxal 5'-phosphate IUBMB (2003): http://www.chem.qmul.ac.uk/iubmb/en- PP diphosphoric acid zyme/reaction/. SAH S-adenosyl-L-homocysteine SENGBUSCH P.: Botany Online –The Internet Hypertext- SAM S-adenosyl-L-methionine book. http://www.biolobie.uni-hamburg.de/b_online/ e00/default.htm. Reference s Received for publication June 13, 2005 AZEVEDO R.A., ARRUDA P., TURNER W.L., LEA P.J. (1997): Accepted after corrections August 13, 2005 The biosynthesis and metabolism of the aspartate de-

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 tel.: + 420 220 443 177, fax: + 420 233 339 990, e-mail: [email protected]

10