Amino Acid Synthesis☆

L Reitzer, The University of Texas at Dallas, Richardson, TX, USA

ã 2014 Elsevier Inc. All rights reserved.

Introduction 2
Amino Acid Requirements and Concentrations, Nitrogen Sources and Global Regulators of Amino Acid
Metabolism 2
Ammonia Assimilation and the a-Ketoglutarate Family: Glutamate, Glutamine, Proline, and Arginine 4
Ammonia Assimilation and Metabolism of Glutamate and Glutamine 4
Regulation of Glutamine and Glutamate Synthesis 5
Proline 5
Arginine 6
The Aspartate Family: Aspartate, Asparagine, Threonine, Lysine, and Methionine 7
Aspartate and Asparagine 7
Threonine, Lysine, and Methionine: Functions and the Common Pathway 7
Threonine 8
Methionine 9
Lysine 9
The Pyruvate Family: Alanine and the Branched-Chain Amino Acids 9
Alanine 9
The Branched-Chain Amino Acids 10
The 3-Phosphoglycerate Family: Serine, Glycine, and Cysteine 11
Serine and Glycine 11
Cysteine 12
The Aromatic Amino Acids 13
Histidine 13

Glossary Feedback inhibition A mechanism of kinetic control by a

Assimilation The incorporation of inorganic compounds, product of a reaction or a pathway that is usually directed at
such as ammonia and sulfate, into organic intermediates. the first enzyme of a pathway.
Attenuation A mechanism of gene expression that senses Nitrogen limitation The slower nitrogen assimilation that
the availability of an amino acid by the extent of tRNA results from utilization of a single nitrogen source other than
charging, which controls the formation of a transcriptional ammonia.
terminator before the first structural gene of an operon. Stringent response A response controlled by guanosine
End-product repression A mechanism of transcriptional tetraphosphate that balances the demand for synthesis of
control in which the product of a reaction sequence proteins and their amino acid precursors.
contributes to the repression of the pathway.

Abbreviations GS Glutamine synthetase

ADP Adenosine diphosphate IMP Inosine monophosphate
ATase Adenylyltransferase Lrp Leucine-responsive regulatory protein
ATP Adenosine triphosphate NAD Nicotinamide adenine dinucleotide
cAMP Cyclic adenosine monophosphate Ntr Nitrogen-regulated
CRP Cyclic-AMP receptor protein PEP Phosphoenolpyruvate
FAD Flavin adenine dinucleotide SAM S-adenosylmethionine
FNR Fumarate nitrate reductase SHMT Serine hydroxy methyltransferase
GCV Glycine cleavage enzyme system UMP Uridine monophosphate
GDH Glutamate dehydrogenase UTase/UR Uridylyltransferase/uridylyl-removing

☆
Change History: January 2014: L Reitzer updated the text and further readings to this entire article, and added Relevant websites.
The following were updated: ‘Keywords,’ ‘Abstract,’ ‘Introduction,’ ‘Amino Acid Requirements and Concentrations, Nitrogen Sources and Global Regulators of
Amino Acid Metabolism,’ ‘Regulation of Glutamine and Glutamate Synthesis,’ ‘Lysine,’ ‘Alanine,’ ‘Further Reading,’ Table 1, and Figures 3–9.

Reference Module in Biomedical Research [Link] 1

2 Amino Acid Synthesis

Introduction

The pathways of amino acid synthesis comprise a significant fraction of a bacterium’s metabolic activity during growth in a
minimal medium. Two amino acids, glutamine and glutamate, are the immediate products of ammonia assimilation and essential
nitrogen donors for the synthesis of other intermediates. Amino acids are not only protein precursors, but also precursors for
numerous other crucial compounds, such as polyamines, S-adenosylmethionine, pantothenic acid, and nucleotides. Our knowl-
edge of the pathways of amino acid metabolism and their regulation is most extensive in the model genetic organism Escherichia
coli. This article focuses on amino acid synthesis in E. coli. Because catabolic pathways may regulate the availability of amino acids,
amino acid degradation is also briefly considered.

Amino Acid Requirements and Concentrations, Nitrogen Sources and Global Regulators of Amino Acid
Metabolism

Figure 1 summarizes the metabolic precursors for amino acid synthesis and provides an overview of the relationships between the
amino acids. Table 1 provides a quantitative description of the amino acid composition of total cellular protein, the use of amino

Figure 1 Amino acid precursors and relationships between amino acids. The thick black arrows indicate the pathways of ammonia assimilation.
 Amino Acid Synthesis 3

Table 1 Amino acid requirementsa

Protein content Precursors for other compounds Nitrogen donation Total Intracellular concentration (mM)
b b
glutamine 250 10 226(2058) 10 476(2308) 3.8
glutamate 250 810 7108 8168 96
aspartate 229 1565 979 2772 4.2
serine 205 1409 1614 0.068
glycine 582 418 1000
alanine 488 55 543 2.6
threonine 241 276 517 0.018
leucine 428 428 0.15
valine 402 402 4.0
lysine 326 28 354 0.40
arginine 281 281 0.57
isoleucine 276 276 0.15
cysteine 87 153 240
asparagine 229 229 0.51
proline 210 210 0.38
phenylalanine 176 176 0.018
methionine 146 7 153 0.14
tyrosine 131 131 0.029
histidine 90 90 0.068
tryptophan 54 54 0.012
a
This table is adapted from Reitzer (2003). Unless indicated, units are mmoles per g dry weight of E. coli grown in glucose-ammonia minimal medium.
b
The first number is for glutamine-dependent glutamate synthesis by the GS-glutamate synthase route. The number in parenthesis is for ammonia-dependent glutamate
synthesis by GDH.

acids as precursors for other compounds, and utilization of amino acids as nitrogen donors for cells grown in a glucose-ammonia
minimal medium, that is, when cells must synthesize all their amino acids. The column labeled ‘Total’ provides an estimate of the
relative rates of the pathways. Amino acids account for about 50% of total intracellular metabolites. Table 1 also shows the absolute
concentrations of amino acids in E. coli using modern technologies.
E. coli can obtain the nitrogen for amino acid synthesis from ammonia, which supports rapid growth. Some bacteria, but not
E. coli, can convert other inorganic nitrogen sources, such as nitrogen gas, to ammonia. E. coli can utilize several organic nitrogen
sources, such as amino acids and nucleosides, as sole nitrogen sources. However, their utilization limits growth, which is
considered nitrogen limited growth. About 100 genes are expressed during nitrogen limitation, and these are referred to as
nitrogen-regulated (Ntr) genes. Their products enhance ammonia assimilation, scavenge nitrogen-containing compounds from
the environment, and integrate various macromolecular processes, such as protein synthesis, with slower metabolism. Nitrogen
limitation affects the regulation of a few enzymes of amino acid synthesis, and several enzymes of amino acid degradation. The
regulators of this global response are described in the section‘Ammonia assimilation and the a-ketoglutarate family: glutamate,
glutamine, proline, and arginine.’
The stringent response balances protein synthesis with the availability of amino acids. When cell growth is limited by a shortage
of a single charged tRNA, guanosine tetraphosphate (ppGpp) accumulates, and stimulates the synthesis of glutamate, glutamine,
the branched-chain amino acids, threonine, methionine, lysine, arginine, histidine, glutamate, and glutamine. In some cases,
ppGpp is an absolute requirement for amino acid synthesis. Mutants devoid of ppGpp are auxotrophic for arginine, all three
branched-chain amino acids, histidine, methionine, phenylalanine, threonine, and, in some strains, glycine and lysine. The
stringent response is also required for ammonia assimilation during nitrogen limitation and for the Ntr response, although the
control is indirect. ppGpp also inhibits nucleotide synthesis, induces nucleoside degradation, and inhibits the synthesis of stable
RNAs (tRNA and rRNA). In sum, the stringent response rapidly balances the relative requirements for amino acids and nucleotides
by simultaneously affecting several interconnected metabolic pathways. The mechanism of ppGpp action is not always known.
Sometimes ppGpp acts through DksA which interacts directly with RNA polymerase. When such regulation has been demon-
strated, it is indicated in the figures.
The leucine-responsive protein (Lrp), which requires ppGpp for its synthesis, controls the expression of several hundred genes.
These genes include those that are required for ammonia assimilation during nitrogen limitation. Control by Lrp is complex. Lrp by
itself may activate or repress expression, and leucine may reverse or enhance this activity.
The availability of carbon and glucose can also affect the expression of many genes of amino acid metabolism, and cyclic-AMP
(cAMP) and its receptor protein (CRP) mediates this control.
The polyamine putrescine alters the expression of several hundred genes, sometimes through the effects on global regulators.
Putrescine appears to block several amino acid biosynthetic pathways, especially those for arginine, histidine, leucine, and
threonine. A rationale for such regulation is not obvious.
4 Amino Acid Synthesis

Ammonia Assimilation and the a-Ketoglutarate Family: Glutamate, Glutamine, Proline, and Arginine
Ammonia Assimilation and Metabolism of Glutamate and Glutamine
The syntheses of glutamine and glutamate are the primary mechanisms for ammonia assimilation. Glutamine synthetase (GS) is
the only enzyme that synthesizes glutamine, but two enzymes synthesize glutamate–glutamate dehydrogenase (GDH) and
glutamate synthase, which aminate a-ketoglutarate with ammonia and glutamine, respectively (Figure 2). Either GS or GDH can
be the major ammonia assimilatory enzyme (Figure 2). Their Kms for ammonia are about 0.1 and 2 mM, respectively.
GDH-dependent ammonia assimilation does not consume energy, but GS-dependent assimilation does. GS is the primary enzyme
of ammonia assimilation when either the ammonia concentration is low, or energy is not limiting (Figure 2(a)). GS also
metabolically traps ammonia, which is important during nitrogen-limited growth, when ammonia can leak through the mem-
brane. Whenever GS is the primary route of ammonia assimilation, glutamate synthase synthesizes glutamate (Figure 2(a)). Cells
without either GS or glutamate synthase fail to utilize most organic nitrogen sources. GDH assimilates ammonia when energy is
limiting (Figure 2(b)). Mutants lacking GDH grow less well in carbon-limited medium.
Glutamate and glutamine are not only the primary products of ammonia assimilation, but they are also the nitrogen donors for
the synthesis of essentially all nitrogen-containing compounds. Glutamate provides approximately 72% of the cell’s nitrogen,
mostly via reversible transaminations, while glutamine provides approximately 28% of the nitrogen for synthesis of histidine,
tryptophan, asparagine, purines, pyrimidines, amino sugars, and p-aminobenzoate. In energy-rich media, glutamine’s amide also
provides the nitrogen for glutamate synthesis.
Glutamate also contributes to other functions. Glutamate is the precursor for proline, arginine, and the polyamine putrescine.
Glutamate decarboxylation is required for resistance to extreme acid (pH  2). The decarboxylation consumes a proton, and the
product, g-aminobutyrate, is exported by an exchange reaction, which brings in another molecule of glutamate. Glutamate is an
osmotically regulated anion. Its concentration varies in parallel with K+. This covariation has at least one known function: K+
accumulation increases the internal pH, which glutamate counterbalances by lowering the pH.
Both glutamate and glutamine can be used as sole nitrogen sources but are not good carbon sources for E. coli. Even though
glutamine is the signal of nitrogen sufficiency (described below), glutamine as a nitrogen source limits growth and induces the Ntr
response. Glutamate synthase may be the most important glutamine catabolic enzyme, although several enzymes with glutaminase

Figure 2 Glutamine and glutamate synthesis, ammonia assimilation, and their control. (a) The reactions with energy excess. (b) The reactions during
energy-limited growth. (c) The regulatory proteins that control GS adenylyiation, GS synthesis, and the Ntr response: solid symbols, nitrogen limitation;
open symbols, nitrogen excess. a-Ketoglutarate antagonizes the actions of PII in nitrogen excess conditions.
 Amino Acid Synthesis 5

activity have also been detected. E. coli does not possess a catabolic GDH. Instead, glutamate might be degraded by transamination-
dependent transfer of the nitrogen to aspartate, and aspartate degradation to fumarate and ammonia.

Regulation of Glutamine and Glutamate Synthesis

Control of glutamine synthesis may be more complex than that for any other enzyme. 40 different metabolic intermediates have
been implicated in its control. Several intermediates exert kinetic control on GS activity. Alanine, glycine, and serine compete with
glutamate at the active site. Compounds that require nitrogen from glutamine–histidine, tryptophan, CTP, AMP, carbamoyl-
phosphate, and glucosamine 6-phosphate – also inhibit GS activity. Each inhibits partially, and together they inhibit more than
each by itself. Such control is an example of cumulative feedback inhibition.
A second layer of kinetic control is covalent adenylylation. GS from nitrogen-replete cells is adenylylated. Adenylylation
inactivates the modified subunit of the GS dodecamer, which reduces GS specific activity. It is not known whether adenylylation
affects feedback inhibition, which was studied before adenylylation was discovered, and has not been reexamined. Glutamine and
a-ketoglutarate regulate a three-protein regulatory cascade that controls GS adenylylation (Figure 2(c)). Glutamine, which senses
nitrogen sufficiency, affects the regulatory cascade at two points. It is the only factor that affects the activity of the bifunctional
uridylyltransferase/uridylyl-removing enzyme (UTase/UR). High glutamine, that is, nitrogen excess, stimulates UMP removal from
the regulatory protein PII (Figure 2(c), open arrows). Unmodified PII interacts with adenylyltransferase (ATase) and stimulates
adenylylation of GS. Glutamine also allosterically stimulates the adenylyl transfer activity of ATase. Low glutamine, the sensor for
nitrogen limitation, stimulates uridylylation of PII, which not only prevents GS adenylylation but also actively promotes dead-
enylylation (Figure 2(c), solid arrows). a-Ketoglutarate, which senses carbon sufficiency, binds to unmodified PII and impairs the
interaction with ATase. It counters the adenylylating effect of glutamine by favoring deadenylylation of GS, but only to the extent
that PII is not modified (i.e., with moderate to high glutamine).
Transcriptional control of GS synthesis involves many of the same regulators that control adenylylation. The complex glnA-
ntrBC operon codes for GS, NtrB, and NtrC. Two minor promoters ensure basal levels of the products of the operon. The minor
promoter preceding glnA requires cyclic-AMP and its binding protein. The major promoter of the operon is utilized during nitrogen
limitation. Low glutamine (nitrogen limitation) results in uridylylation of PII, which is unable to interact with NtrB (Figure 2(c),
solid arrows). In this situation, NtrB phosphorylates NtrC, which interacts with a s54-containing RNA polymerase and stimulates
transcription of glnA and other Ntr genes. High glutamine (nitrogen excess) results in the net dephosphorylation of NtrC-P
(Figure 2(c), open arrows). Glutamate synthase is required for induction of the Ntr response. In its absence, either glutamine
accumulates, which prevents Ntr induction, or there is insufficient glutamate for new protein synthesis. The regulators of ammonia
assimilation are in turn controlled by PurR, Fnr, and Fur, which sense purines, fumarate/nitrate, and iron, respectively.
GS specific activity, glnA expression, and Ntr gene expression vary in parallel (Figure 2(c)). However, they do not respond
identically to changes in glutamine concentration. The adenylylation system senses glutamine at a higher concentration range, and
it also responds to smaller changes in glutamine concentration. Therefore, the first response to nitrogen limitation is to increase the
specific activity of the available GS, and the second response is to affect gene expression. Adenylylation is also important in the
transition from a nitrogen-poor to a nitrogen-rich environment since it prevents depletion of the glutamate pool.
Much less is known about the regulation of glutamate synthesis. Aspartate appears to be a major kinetic inhibitor of glutamate
synthase. Glutamate represses both GDH and glutamate synthase, but in each case, the mechanism of repression is unknown.
Carbon limitation, presumably via cAMP-CRP, moderately represses both enzymes. The leucine-responsive protein (Lrp) is
required for the synthesis of glutamate synthase. ArgP, ArgR, Fnr, Fur, and Nac have also been implicated in control of these
enzymes. These regulators sense lysine, arginine, fumarate/nitrate, iron availability, and nitrogen limitation, respectively.

Proline
Proline is not a precursor for other metabolites. However, at high environmental osmolality, proline serves to control cellular
osmolality.
The first two enzymes of proline synthesis form a complex which phosphorylates and reduces glutamate to form g-glutamic
semialdehyde, which spontaneously cyclizes to L-D1-pyrroline-5-carboxylate (Figure 3, top line). The third enzyme reduces this
intermediate to proline. Both reductions require NADPH. The only control on this pathway is feedback inhibition of g-glutamyl
kinase by proline. The genes appear to be constitutively expressed. High osmolality, which results in proline accumulation, inhibits
proline catabolism and enhances proline transport but is not known to affect proline synthesis.
The enzymes of arginine synthesis provide an alternate route for formation of g-glutamic semialdehyde (Figure 3, dashed
arrow). A block in the fourth step of arginine biosynthesis results in accumulation of N-acetylglutamic g-semialdehyde, which can
be deacetylated by the fifth enzyme of arginine synthesis to form g-glutamic semialdehyde.
The catabolic pathway is a reversal of the anabolic pathway, except that the electron acceptor is FAD for the first oxidation. The
electrons from FADH2 are donated to the electron transport chain, and possibly accounts for the membrane association of the
catabolic enzyme. A single protein, PutA, catalyzes the three reactions of proline catabolism. PutA is also a transcriptional regulator
that controls its own synthesis. Proline and oxygen are required for expression.
6 Amino Acid Synthesis

Figure 3 Proline and arginine synthesis. Effectors of enzyme activity and cofactor requirements are under the pathways. Inhibitory compounds are in
parentheses. Compounds that antagonize inhibitors, essentially activators, are in brackets. Effectors of transcriptional control are above pathways.
Repressors are in parentheses, while activators are not. (A) indicates repression by ArgR complexed with arginine. arg, arginine; GLT, glutamate; aKG,
a-ketoglutarate; pro, proline; PxP, pyridoxal phosphate.

Arginine
Either arginine or ornithine, an intermediate in arginine synthesis, can be used to synthesize the polyamines putrescine and
spermidine. Ornithine is also a precursor for the hydroxamate type of iron siderophores. Another arginine precursor, carbamoyl
phosphate, is an essential precursor for pyrimidines.
The first five reactions of arginine synthesis add an amino group to the d-carbon of glutamate to form ornithine (Figure 3,
middle line). The first reaction acetylates glutamate’s amino group, which prevents subsequent cyclization, and can occur by two
distinct mechanisms: direct acetylation by acetyl-CoA (as in E. coli) or transacetylation with N-acetylornithine, which is also an
intermediate in arginine synthesis. The latter mechanism appears to be more common in bacteria. Phosphorylation, reduction, and
addition of an amino group by transamination produce N-acetylornithine. The acetyl group is then removed either by deacetyla-
tion, as in E. coli, or by transacetylation, which recycles the acetyl group back to glutamate and forms the first intermediate of the
pathway. Ornithine combines with carbamoyl phosphate, which requires glutamine as the nitrogen donor, to form citrulline
(Figure 3, bottom line). The last two steps of arginine synthesis amount to donation of aspartate’s nitrogen to citrulline.
The two major sites of kinetic control are at the beginning of the pathway leading to ornithine and at the branch point where
carbamoyl phosphate enters the biosynthetic pathway. Arginine inhibits the enzyme that acetylates glutamate. UMP inhibits
carbamoyl phosphate synthetase, and ornithine and IMP prevent this inhibition. In addition, orotate, an intermediate in
pyrimidine synthesis, activates ornithine transcarbamoylase. These mechanisms balance the relative need for arginine and
pyrimidines. Some organisms, such as Bacillus subtilis, have two carbamoyl phosphate synthetases – one for pyrimidines and the
other for arginine.
The major mode of transcriptional regulation in E. coli is repression by ArgR, which binds operator sites when bound with
arginine. In E. coli, many of the arginine biosynthetic genes are not linked. ArgR represses each operon to a differing extent. The gene
for carbamoyl phosphate synthetase has two promoters, and is regulated by several protein complexes. These include ArgR, which
responds to arginine; a complex of PurR-IHF-PepA, which responds to purines; and a complex of PyrH-IHF-PepA, which responds
to pyrimidines. In addition to its function as a repressor, ArgR also resolves plasmid dimers to monomers, which is required for
maintaining copy number. ppGpp increases the levels of the first and fifth enzymes of arginine synthesis. The mechanism of
activation is unusual for the first enzyme – it appears that ppGpp decreases errors during transcription. Finally, cAMP-CRP activates
the expression of the penultimate enzyme of arginine synthesis.
E. coli can degrade arginine as a nitrogen source, but not as a carbon source. The catabolic pathway is reminiscent of the anabolic
pathway. Instead of acetylation, the amino group of arginine is succinylated. The two nitrogens and carbon added in the last three
steps of arginine synthesis are removed in one step to form succinylornithine. The subsequent reactions deaminate, oxidize, and
 Amino Acid Synthesis 7

desuccinylate, resulting in formation of glutamate. This pathway is induced by nitrogen limitation and entry into stationary phase.
Arginine and ornithine decarboxylases have been thoroughly studied, but do not contribute to nitrogen source utilization. Instead,
they primarily contribute to survival in acidic, anaerobic environments.

The Aspartate Family: Aspartate, Asparagine, Threonine, Lysine, and Methionine

Aspartate and Asparagine
Aspartate is the precursor for threonine, methionine, lysine, pyrimidines, pantothenate, and NAD. Aspartate is a nitrogen donor for
arginine synthesis and for two reactions in purine synthesis. Aspartate, and not oxaloacetate or malate, is a metabolic sensor of
dicarboxylic acid availability. High aspartate inhibits dicarboxylic acid formation from PEP and also stimulates enzymes that
convert dicarboxylic acids to PEP and pyruvate. In contrast, asparagine is a dead-end metabolite.
Glutamate-dependent transamination of oxaloacetate produces aspartate (Figure 4). Two enzymes can catalyze this reaction.
Aspartate transaminase, specified by aspC, is quantitatively more important, and its synthesis is constitutive. The tyrosine-
phenylalanine transaminase, the tyrB product, can also synthesize aspartate, and tyrosine represses its synthesis. Aspartate
transaminase activity is very high, which suggests that its products and substrates are in equilibrium, and that mass action controls
aspartate synthesis. Considering the numerous functions of aspartate, such regulation is appropriate.
Ammonia- and glutamine-dependent asparagine synthetases (AsnA and AsnB, respectively) catalyze the ATP-dependent
amidation of aspartate with release of pyrophosphate (Figure 4). The ammonia-dependent enzyme is present in prokaryotes,
but not eukaryotes. AsnB is required for growth in a nitrogen-limited medium, presumably because of insufficient ammonia for
AsnA. AsnB has a low catalytic capacity, and becomes 5% of the soluble protein during nitrogen-limited growth. When ammonia is
available, AsnA presumably spares the need to synthesize the inefficient AsnB.
Asparagine inhibits and represses both enzymes. In the absence of asparagine, the two enzymes are reciprocally regulated.
Nitrogen limitation represses AsnA. Two regulators control the formation of AsnA. In E. coli, AsnC activates and asparagine
antagonizes this activation; in Klebsiella pneumoniae (and probably E. coli), Nac, a regulator induced by nitrogen limitation,
represses. Regulators that control AsnB synthesis have not been studied, although a mechanism that mediates repression by
asparagine would be sufficient to account for all known regulation.
For E. coli, aspartate and asparagine are degraded slowly as carbon sources, and both are good nitrogen sources. Both amino
acids are sources of fumarate, which can be used as a terminal electron acceptor during anaerobic respiration. The most likely
aspartate catabolic enzyme is aspartase, which generates fumarate and ammonia. Glucose represses aspartase formation, presum-
ably via cyclic-AMP and its binding protein, and FNR activates during anaerobic growth. Asparaginases degrade asparagine to
aspartate and ammonia. E. coli contains a high-affinity periplasmic asparaginase, and a low-affinity cytoplasmic asparaginase. The
cytoplasmic asparaginase appears to be required for utilization of asparagine as carbon or nitrogen source, but nothing is known
about its regulation. The regulation of the periplasmic asparaginase is similar to that of aspartase – glucose represses and FNR
activates during anaerobic growth.

Threonine, Lysine, and Methionine: Functions and the Common Pathway

Threonine is the precursor for isoleucine, and in unusual circumstances it can be the precursor for glycine and serine. Diamino-
pimelate, an intermediate in lysine synthesis, is an essential component of peptidoglycan. Lysine can be decarboxylated to form
cadaverine, which can replace putrescine as the major polyamine. Numerous secondary metabolites are derived from intermediates

Figure 4 Aspartate and asparagine synthesis. Inhibitors of enzyme activity are shown below the pathway in parentheses. Effectors of transcriptional
control are above pathways. Repressors are in parentheses, while activators are not. AsnC/(asn) indicates that AsnC is an activator and asparagine (asn)
prevents this activation. asn, asparagine; GLN, glutamine; GLT, glutamate; aKG, a-ketoglutarate; PPi, inorganic pyrophosphate; tyr, tyrosine.
8 Amino Acid Synthesis

in lysine synthesis in several organisms. Methionine is the precursor of S-adenosylmethionine (SAM), a donor for protein and
nucleic acid methylations, for propylamino groups of polyamines, and for cyclopentenediol for tRNA modifications.
The synthesis of these amino acids involves a single common pathway and three amino acid-specific pathways (Figure 5). The
common pathway results in the synthesis of homoserine and starts with the phosphorylation of aspartate by aspartokinase, which
produces b-aspartyl phosphate (Figure 5, top line). Two NADPH-dependent reductions, catalyzed by aspartic semialdehyde
dehydrogenase and homoserine dehydrogenase, convert b-aspartyl phosphate to aspartate semialdehyde (the precursor for lysine)
and then to homoserine (the precursor for threonine and methionine), respectively. E. coli has three aspartate kinases and two
homoserine dehydrogenases – the first and third reactions of the common pathway, respectively. The homoserine dehydrogenases
are part of the same polypeptide that contains aspartokinase. These isozymes are subject to different patterns of kinetic and
transcriptional control, which ensures that an excess of one amino acid does not prevent synthesis of the others (Figure 5).

Threonine
Threonine is derived in two steps from homoserine (Figure 5, top line). Homoserine is phosphorylated, and threonine synthase
catalyzes a complex reaction that converts the resulting homoserine phosphate to threonine. Aspartokinase-homoserine dehydro-
genase I is the major homoserine dehydrogenase activity. Threonine allosterically inhibits its activity. Serine also inhibits this
enzyme, and this is the basis for serine toxicity in some strains. Threonine also inhibits the first enzyme specific to threonine
synthesis, homoserine kinase. The thrABC operon codes for aspartokinase-homoserine dehydrogenase I and the two enzymes of the
threonine-specific pathway, respectively. Threonine and isoleucine, which is derived from threonine, control its expression through
an attenuation mechanism.

Figure 5 Threonine, lysine and methionine synthesis. Effectors of enzyme activity and cofactor requirements are under the pathways. Inhibitory
compounds are in parentheses. Effectors of transcriptional control are above pathways. Repressors are in parentheses, while activators are not.
A protein designation followed by a compound in parenthesis, for example, ArgP/(lys), means that the protein is a transcriptional activator and the
compound prevents this activation. Attenuation is indicated by tRNA and an amino acid. B12, vitamin B12; isl, isoleucine; GLT, glutamate; HC,
homocysteine; aKG, a-ketoglutarate; leu, leucine; lys, lysine; phe, phenylalanine; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PxP, pyridoxal
phosphate; SAM, S-adenosylmethionine; ser, serine; THF, tetrahydrofolate; thr, threonine.
 Amino Acid Synthesis 9

Several enzymes can degrade threonine – an aldolase, a dehydrogenase, and a deaminase. Threonine aldolase generates
acetaldehyde and glycine. Its function is not certain. The dehydrogenase pathway degrades threonine in two steps to acetyl-CoA
and glycine. Acetyl-CoA is required for carbon source utilization, whereas glycine and its subsequent degradation are required for
nitrogen source utilization. Lrp activates expression of its genes. The deaminase/dehydratase pathway is an anaerobic pathway that
produces propionyl-phosphate, which generates ATP by a substrate level phosphorylation. Its genes are under tight catabolite
repression.

Methionine
Methionine is synthesized from homoserine in four steps (Figure 5, third line). Three complex reactions replace the hydroxyl group
of homoserine with -SH, which produces homocysteine. Succinylation of the hydroxyl group by succinyl-CoA initiates these
reactions in E. coli, whereas acetylation is employed in gram-positive organisms. Cysteine is added in the second reaction and is the
source of the reduced sulfur. The third enzyme of the pathway hydrolyzes the intermediate cystathionine. This enzyme can also
hydrolyze cysteine and serine. Serine inhibits this reaction and exogenous serine can result in a methionine requirement. In the
fourth reaction, donation of a methyl group to the sulfur of homocysteine generates methionine. The methyl donor is
N5-methyltetrahydrofolate, which is generated by reduction of N5, N10-methylene tetrahydrofolate (Figure 5, bottom line). Two
methionine synthases catalyze the final methyl transfer to homocysteine: One requires cobalamin (vitamin B12), whereas the other
does not. The latter is sensitive to oxidative stress. S-adenosylmethionine (SAM) is synthesized in one step from methionine
and ATP.
The only kinetic control of methionine synthesis is inhibition of the first methionine-specific enzyme, homoserine transsucci-
nylase, by methionine or SAM. Unlike the kinetic control, the transcriptional controls are surprisingly complex. The genes of
methionine synthesis are generally unlinked. The repressor MetJ bound with SAM, a product of methionine metabolism, represses
aspartokinase-homoserine dehydrogenase II of the common pathway and all the methionine-specific enzymes, except the
cobalamin-dependent methionine synthase. MetR is a second regulator of the met regulon. MetR, which binds homocysteine,
can either inhibit or stimulate transcription. MetR’s most noticeable effect is the activation of the genes for the two methionine
synthases. (MetJ and MetR also regulate glyA, which codes for serine hydroxy methyltransferase, an enzyme involved in synthesis of
N5, N10-methylene tetrahydrofolate, which is required for methionine synthesis.) In addition, cobalamin with MetH (the
cobalamin-dependent methionine synthase) can repress or activate, but the mechanism of regulation has not been established.
Homoserine transsuccinylase (encoded by metA) is thermolabile, and at high temperatures metA expression requires s32, which
means that metA is a heat shock gene.

Lysine
Lysine synthesis starts with condensation of aspartate semialdehyde and pyruvate to form a cyclic intermediate, which is reduced by
NADPH to form tetrahydrodipicolinate (Figure 5, second line). In E. coli, tetrahydrodipicolinate is succinylated by succinyl-CoA,
nitrogen is added by glutamate-dependent transamination, and the blocking group is removed to produce LL-diaminopimelate.
An epimerase converts LL-diaminopimelate to meso-diaminopimelate, which is decarboxylated to form lysine. In some organisms,
tetrahydrodipicolinate is converted directly to meso-diaminopimelate, and in gram-positive organisms the blocking agent is an
acetyl group. The only known transaminase is also the fourth enzyme of arginine synthesis. Deletion of the gene for this enzyme
has no effect on growth, which suggests redundant enzymes for the transaminase reaction.
The regulation of lysine synthesis is complex, which is appropriate considering the number of important intermediates that are
synthesized. Lysine kinetically controls aspartokinase III and the first committed reaction of lysine synthesis – the condensation of
pyruvate with aspartic semialdehyde. The genes of the lysine-specific pathway are not linked. ArgP activates transcription of five of
the nine enzymes that convert aspartate to lysine, and lysine prevents this activation. LysR complexed with diaminopimelate
activates transcription of diaminopimelate decarboxylase, which is the last enzyme of the pathway. Diaminopimelate starvation
regulates the first committed step of lysine synthesis by an unknown mechanism.

The Pyruvate Family: Alanine and the Branched-Chain Amino Acids

Alanine
L-Alanine is the precursor for D-alanine, which is a major component of the cell wall. L-Alanine also binds to Lrp, a global
regulator, and affects its activity. L-Alanine auxotrophs have only recently been isolated. Three major transaminases can synthesize
L-alanine from pyruvate: AvtA, AlaA, and AlaC (Figure 6). However, eight other transaminases can synthesize L-alanine when
overproduced or with pyruvate as the carbon source. The amino donor for all of these enzymes, except for AvtA, is glutamate; the
amino donor for AvtA is valine. Old labeling studies suggested a pyruvate-independent pathway for L-alanine synthesis, but the
recent genetic analysis showed that pyruvate is the precursor for almost all L-alanine synthesis. D-Alanine is synthesized from
L-alanine by a constitutive racemase.
10 Amino Acid Synthesis

Figure 6 Branched-chain amino acid synthesis. Effectors of enzyme activity and cofactor requirements are under the pathways. Inhibitory compounds
are in parentheses. Compounds that antagonize inhibitors, essentially activators, are in brackets. Effectors of transcriptional control are above pathways.
Repressors are in parentheses, while activators are not. Attenuation is indicated by tRNA and the amino acid. ala, alanine; bcaa, the sum of leucine,
isoleucine, and valine; GLT, glutamate; isl, isoleucine; aKG, a-ketoglutarate; leu, leucine; PxP, pyridoxal phosphate; TPP, thiamine pyrophosphate;
tyr, tryosine; X, a-aceto-a-hydroxybutyrate; Y, a-acetolactate.

L- and D-Alanine can be used as nitrogen or carbon sources. L-Alanine is racemized to D-alanine, which is degraded to pyruvate
and ammonia by the non-specific membrane-bound D-amino acid dehydrogenase. Expression of the dehydrogenase requires
L-alanine, cyclic-AMP and its binding protein, and Lrp.

The Branched-Chain Amino Acids

Isoleucine, valine, and leucine are not precursors for other intermediates. However, the penultimate intermediate for valine,
a-ketoisovalerate, is also a precursor for pantothenate and leucine. High a-ketobutyrate, which is an intermediate of isoleucine
synthesis, is toxic and has been proposed to be an alarmone. Proteins have only a limited ability to distinguish between the
structurally related, hydrophobic, branched-chain amino acids and intermediates that lead to their synthesis. The biosynthetic
pathways take advantage of these properties. The aminoacyl-tRNA synthetases are the only proteins that can discriminate between
the branched-chain amino acids, and elaborate mechanisms reduce the frequency of mischarging.
Isoleucine synthesis starts with deamination of threonine, which produces a-ketobutyrate (Figure 6, top line). Pyruvate, which
has one less carbon, is the corresponding a-keto acid for valine synthesis (Figure 6, middle line). Parallel pathways with shared
enzymes then convert a-ketobutyrate and pyruvate to isoleucine and valine, respectively. Acetohydroxy acid synthase catalyzes the
decarboxylation of pyruvate and transfer of acetaldehyde to either a-ketobutyrate or pyruvate. E. coli contains three isozymes of this
enzyme, which differ in substrate specificity and sensitivity to feedback inhibitors. Isozyme I and II preferentially synthesize valine
and isoleucine, respectively. Isozyme I is important for growth with a poor carbon source, whereas isozymes II and III are used only
for growth with glucose. This reaction creates a branch point at the a-carbon. The next enzyme reduces a ketone to an alcohol and
 Amino Acid Synthesis 11

transfers the branch point to the b-carbon. Dehydration and transaminase-dependent amination result in formation of isoleucine
and valine.
Leucine is one methylene group larger than valine and has a branch point at the g-carbon. The extra carbon is added by reactions
similar to those catalyzed by three reactions of the citric acid cycle, which have the net effect of adding a two-carbon unit (acetate),
followed by a decarboxylation (Figure 6, bottom line). The first intermediate in leucine synthesis is a-ketoisovalerate, which is also
an intermediate in valine synthesis. a-Ketoisovalerate is acetylated with acetyl-CoA, and the product, a-isopropylmalate, is
converted to b-isopropylmalate by a reaction similar to that of aconitase. b-Isopropylmalate is then decarboxylated, forming
a-ketoisocaproate, which acquires a nitrogen by transamination to produce leucine.
Allosteric regulation controls the first committed step for each amino acid, but the allosteric regulation is complicated because
of the metabolic interrelationships and the shared enzymes. Isoleucine inhibits threonine deaminase, which is an isoleucine-
specific enzyme. Valine reverses this inhibition. Valine inhibits isozymes I and III of acetohydroxy acid synthase. This can result in
valine toxicity, because of failure to synthesize isoleucine and toxic accumulation of a-ketobutyrate. To prevent this toxicity, several
enteric eubacteria contain a valine-insensitive acetohydroxy acid synthase, isozyme II. E. coli lacks this isozyme and apparently
escapes valine toxicity because of an acetohydroxy acid synthase isozyme that prefers a-ketobutyrate over pyruvate. The major
allosteric control of leucine synthesis is leucine inhibition of isopropylmalate synthase, which catalyzes the first committed step of
leucine synthesis.
Several mechanisms transcriptionally regulate the formation of branched-chain amino acid enzymes. Attenuation controls the
expression of 11 of the 15 genes – the effectors are shown above the pathways in Figure 5. Lrp induces one acetohydroxy acid
synthase isozyme and the leuABCD operon, which codes for the leucine biosynthetic enzymes. In contrast to these mechanisms of
regulation, IlvY activates only one gene, ilvC, which specifies the third enzyme of isoleucine synthesis and the second enzyme of
valine synthesis. IlvY binds the acetohydroxy acids that are the substrates for IlvC. A final layer of regulation is activation by ppGpp
(Figure 6).

The 3-Phosphoglycerate Family: Serine, Glycine, and Cysteine

Serine and Glycine
Serine is a precursor for cysteine, selenocysteine, tryptophan, glycine, and phospholipids. Glycine is a precursor for purines,
pyridoxal, and heme-containing compounds. Glycine synthesis and cleavage generate C1 units, which are required for the synthesis
of purines, thymine, methionine and pantothenate, and the formylation of the initiator tRNAMet. The serine-glycine pathway has
been estimated to account for about 15% of the carbon assimilated by glucose-grown cells. Serine and glycine inhibit glutamine
synthetase. The rationale for such regulation probably involves purine synthesis. Purine synthesis requires serine, glycine, C1 units
and glutamine. High serine and glycine may indicate purine sufficiency, and a diminished need for glutamine for purine synthesis.
Almost half the glutamine synthesized is used for purine synthesis, if glutamine is not used for glutamate synthesis. Serine also
inhibits homoserine dehydrogenase I and threonine deaminase, which are required for isoleucine synthesis, and the third enzyme
of methionine synthesis.
NAD-dependent oxidation of the glycolytic intermediate 3-phosphoglycerate initiates the major pathway of serine synthesis
(Figure 7). Nitrogen is added to the resulting product, 3-phosphohydroxypyruvate, by glutamate-dependent transamination, thus
forming 3-phosphoserine. Dephosphorylation of 3-phosphoserine then produces serine. Serine hydroxy methyltransferase
(SHMT) catalyzes the reversible conversion of serine to glycine and the formation of the C1 carrier N5, N10-methylene tetrahy-
drofolate from tetrahydrofolate. The oxidative cleavage of glycine by the glycine cleavage enzyme system (GCV) produces a second
molecule of N5, N10-methylene tetrahydrofolate, as well as ammonia and CO2. This enzyme may seem unnecessary, but mutants
deficient in GCV excrete glycine, which implies that it is active. GCV is a complex of four different polypeptides.
Mutants deficient in SHMT require glycine, which implies that 3-phosphoglycerate is the major source of glycine. The
dehydrogenase pathway of threonine degradation also generates serine and glycine. Threonine is degraded in two steps to acetyl-
CoA and glycine (Figure 7, second line). Serine is generated from the combined actions of GCV, which produces a C1 unit, and a
reversal of the SHMT reaction, which consumes the C1 unit (Figure 7, top line). This pathway is active only during carbon-limited
growth in the presence of all three branched-chain amino acids and arginine. The former probably increases intracellular threonine,
while the function of arginine is not apparent.
Serine inhibits the activities of several enzymes, which suggests that the intracellular concentration of serine is tightly regulated.
Serine allosterically inhibits 3-phosphoglycerate dehydrogenase, the first enzyme of the major serine pathway. Serine, glycine, or
the products of C1 metabolism do not affect the activity of any other enzyme of this pathway. In contrast, the transcriptional
regulation is complex and only partially understood. C1 sufficiency is sensed by a balance of homocysteine to S-adenosylmethio-
nine. These sensors control SHMT synthesis through MetR, an activator that binds homocysteine (a sensor of C1 deficiency), and
MetJ, a repressor that binds S-adenosylmethionine (a sensor of C1 excess) and controls MetR synthesis. Other products that require
C1 units also repress enzymes of this pathway. The purines hypoxanthine and guanine bind PurR, which then represses SHMT and
GCV. A complex of GcvA-GcvR represses GCV synthesis. Glycine causes dissociation of GcvR, and a GcvA-glycine complex activates
transcription. CRP-cAMP may also reverse repression by GcvA-GcvR. In addition to these regulators, the leucine-responsive protein,
Lrp, also controls these genes. Lrp in the absence of leucine tends to favor the primary pathway of serine and glycine synthesis. Lrp
with leucine decreases the primary pathway, increases the secondary serine synthesis pathway, that is, the threonine dehydrogenase
12 Amino Acid Synthesis

Figure 7 Synthesis of serine, glycine and cysteine and sulfate assimilation. Effectors of enzyme activity and cofactor requirements are under the
pathways. Inhibitory compounds are in parentheses. Effectors of transcriptional control are above pathways. Repressors are in parentheses, while
activators are not. Lrp/(leu) indicates that Lrp is a transcriptional activator, and leu prevents this activation. < > indicates compounds required
for stability. C1-THF, N5,N10-methylene-tetrahydrofolate; GLT, glutamate; aKG, a-ketoglutarate; NAS, N-acetylserine; PPi, inorganic pyrophosphate;
PxP, pyridoxal phosphate; THF, tetrahydrofolate.

pathway of threonine catabolism, and increases serine catabolism. Finally, nitrogen limitation and Ntr response regulators repress
3-phosphoglycerate dehydrogenase, which presumably lowers serine concentration and prevents serine inhibition of glutamine
synthetase when its primary function is ammonia assimilation.
Because of serine toxicity, degradative enzymes might contribute to maintaining the intracellular serine concentration. The
primary enzymes of serine catabolism are serine deaminases/dehydratases. E. coli contains three distinct serine deaminases, and
three other enzymes have serine deaminase activity as a secondary reaction. The regulation of these enzymes is astonishingly
complex. Without going into detail, it is noted that serine can be degraded as sole carbon source, but only in the presence of leucine
or glycine, which is required for induction of catabolic enzymes. Glycine can be utilized as sole nitrogen source. The pathway
involves GCV, formation of serine by SHMT, and subsequent catabolism of serine.

Cysteine
The synthesis of cysteine assimilates inorganic sulfur (Figure 7). Cysteine is a precursor for glutathione, and it donates its sulfur for
the synthesis of methionine and several cofactors. The predominant mechanism of cysteine synthesis consists of two converging
pathways. One branch reduces sulfate, the most abundant environmental source of sulfur, to sulfide. Sulfate is initially activated by
formation of the mixed anhydride, adenosine 5’-phosphosulfate, in a reaction that appears to be driven by GTP hydrolysis
(Figure 7, bottom line). Thioredoxin or glutaredoxin reduce the sulfate moiety to sulfite, which is then reduced to sulfide by
NADPH. NADPH-dependent sulfite reductase contains siroheme, a rare form of heme. The first reaction of the second branch of
the pathway is catalyzed by serine transacetylase, which produces O-acetylserine from acetyl-CoA and serine (Figure 7, third line).
Sulfide reacts with O-acetylserine to form cysteine. An alternate pathway involves thiosulfate as the source of sulfur. Thiosulfate can
condense with O-acetylserine, forming S-sulfocysteine, which is proposed to be reduced to cysteine. This pathway may be required
for the anaerobic synthesis of cysteine.
 Amino Acid Synthesis 13

Excess cysteine is toxic, in part because it appears to inhibit many of the same enzymes that serine inhibits. Therefore, sulfur
incorporation is tightly regulated. A major point of control is cysteine inhibition of serine transacetylase. Allosteric control does not
affect any other enzyme of either branch. However, the activities of the first two enzymes of sulfate reduction rapidly decay with low
O-acetylserine, which prevents sulfate activation when cysteine is available.
Seven operons contain the 15 structural genes that are required for cysteine synthesis and the transport of sulfate and thiosulfate.
CysB, an activator of the LysR family, controls the expression of 13 of these genes. N-acetylserine is the inducer that binds CysB.
N-acetylserine is believed to form spontaneously from O-acetylserine (Figure 7). Sulfide and thiosulfate are anti-inducers which
bind to CysB and block the effect of N-acetylserine binding. These regulatory mechanisms provide a hierarchy of sulfur sources that
determine the extent of induction. Cysteine (assimilated sulfur) is the best sulfur source, and it prevents inducer synthesis. Sulfate,
sulfite, and thiosulfate are moderately good sulfur sources that cause high levels of anti-inducers. Glutathione, a poor sulfur source,
does not prevent inducer synthesis or the binding of inducer to CysB, and is therefore the most derepressing sulfur source.
Cysteine desulfhydrase degrades cysteine to pyruvate, ammonia, and sulfide. E. coli does not contain a dedicated cysteine
desulfhydrase. Instead, desulfhydrase activity is a secondary activity of tryptophanase and MetC, the third enzyme of methionine
synthesis. In contrast, S. typhimurium contains a dedicated cysteine desulfhydrase.

The Aromatic Amino Acids

Phenylalanine and tryptophan are not the precursors for other intermediates, whereas tyrosine donates carbon and nitrogen for the
thiazole unit of thiamine. Chorismate, the last common precursor for all three aromatic amino acids, is also a precursor for
ubiquinone, menaquinone, folate, p-aminobenzoate, and enterochelin (a phenolic iron-binding siderophore).
The first seven steps in the aromatic amino acid pathway, sometimes called the shikimate pathway (after the first identified
intermediate), synthesizes chorismate, which is a precursor for all three aromatic amino acids (Figure 8, top line). The 10 carbons
of chorismate are derived from erythrose 4-phosphate and two molecules of phosphoenolpyruvate. Three isozymes catalyze the
first reaction, and they are the target of kinetic control. A combination of phenylalanine and tyrosine regulates the activities of the
two most active isozymes, whereas tryptophan controls the activity of the third. In each case, the inhibition is incomplete, which
permits synthesis of chorismate, a precursor for several other compounds.
The next two steps of phenylalanine and tyrosine synthesis involve different bifunctional enzymes (Figure 8, second and third
lines). The phenylalanine enzyme is a chorismate mutase-prephenate dehydratase that generates phenylpyruvate, which is
transaminated to form phenylalanine. The tyrosine enzyme is a chorismate mutase-prephenate dehydrogenase that produces
4-hydroxyphenylpyruvate, which forms tyrosine after transamination (Figure 8). Phenylalanine and tyrosine also inhibit the
second reactions of their specific pathways after chorismate, that is, the dehydratase or dehydrogenase. The primary transaminase
in both phenylalanine and tyrosine synthesis is TyrB, although aspartate transaminase, AspC, can also synthesize tyrosine and
phenylalanine. The transaminase for branched-chain amino acids, IlvE, can generate phenylalanine. Tryptophan formation from
chorismate requires phosphoribosylpyrophosphate, serine, and glutamine as a nitrogen donor (Figure 8, bottom line). Trypto-
phan inhibits anthranilate synthetase, the first reaction specific for tryptophan synthesis, and anthranilate inhibits the fourth
reaction.
The transcriptional control of the aromatic amino acid pathways is mediated by TyrR (sometimes complexed with tyrosine or
tryptophan), TrpR (complexed with tryptophan), and tRNA-mediated attenuation. TyrR-dependent regulation may require a
specific corepressor. TyrR represses two of the isozymes of the first step in the shikimate pathway, and TrpR represses the third.
Most enzymes of the shikimate pathway are produced constitutively. This constitutivity contrasts with the regulation of all enzymes
of the specific pathways, which are end-product repressed. Phenylalanine-mediated attenuation controls expression of the first gene
of the phenylalanine pathway. TyrR controls the synthesis of the first gene of the tyrosine pathway (which codes for a bifunctional
protein) and of tyrB, which codes for the phenylalanine–tyrosine transaminase. Finally, the well-studied trp operon is controlled by
a combination of repression by TrpR and tryptophan-mediated attenuation.
E. coli can catabolize tryptophan as a carbon source. The enzyme tryptophanase degrades tryptophan to indole, pyruvate, and
ammonia. Tryptophanase also degrades serine and cysteine. Its expression is controlled by catabolite repression, that is, cAMP-CRP,
and tryptophan-specific induction. Tryptophan controls an unusual attenuation mechanism – tryptophan is required for tran-
scription past a terminator located in front of the structural gene. A variety of other environmental factors, such as growth in
alkaline medium, induces tryptophanase synthesis. Indole, a product of the tryptophanase reaction, regulates the expression of
several genes by an unknown mechanism. Indole production is one of the properties used to distinguish E. coli from other bacteria.

Histidine

Histidine is not a precursor for other metabolic intermediates, and, with only one exception, intermediates in histidine synthesis
are only required for histidine synthesis. Histidine is synthesized in nine steps (Figure 9). The first reaction is condensation of
phosphoribosyl pyrophosphate and ATP, which provides all the carbon atoms. A glutamine-dependent amidotransferase and a
glutamate-dependent transaminase provide the nitrogens required for the pathway. A curious feature of the pathway is the release
of 5-aminoimidazole-4-carboxamide ribonucleotide, an intermediate in purine synthesis, which links histidine and purine
14 Amino Acid Synthesis

Figure 8 Aromatic amino acid synthesis. Effectors of enzyme activity and cofactor requirements are under the pathways. Inhibitory compounds are in
parentheses. Strong inhibitors are shown with solid arrows, while weak inhibitors with dashed arrows. Effectors of transcriptional control are above
pathways. Repressors are in parentheses, while activators are not. Attenuation is indicated by tRNA and an amino acid. bcaa, the sum of leucine,
isoleucine, and valine; GLN, glutamine; GLT, glutamate; aKG, a-ketoglutarate; PEP, phosphoenolpyruvate; phe, phenylalanine; Pi, inorganic phosphate;
PPi, inorganic pyrophosphate; PRPP, phosphoribosylpyrophosphate; PxP, pyridoxal phosphate; trp, tryptophan; tyr, tyrosine.

Figure 9 Histidine synthesis. Inhibitors of enzyme activity are shown below the pathway in parentheses. The genes for all enzymes of this pathway
form an operon, and histidine represses the operon by an attenuation mechanism. GLN, glutamine; GLT, glutamate; his, histidine; aKG,
a-ketoglutarate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PRPP, phosphoribosylpyrophosphate.
 Amino Acid Synthesis 15

synthesis. Failure to recycle this intermediate results in a significant decrease in purine pools and synthesis. Furthermore, over-
production of the pathway results in an adenine auxotrophy.
Kinetic control of the pathway is directed at the first enzyme. Histidine, ppGpp, and two indicators of a low energy status (ADP
and AMP) inhibit this reaction. Histidine is required for the inhibition by ppGpp. Histidine also enhances the allosteric binding of
ADP and AMP. Transcriptional control is facilitated by organization of the eight genes of histidine synthesis into a single operon.
ppGpp positively controls his operon expression and accounts for a 30-fold range of expression. An attenuation mechanism that
senses histidine deficiency by the charging of tRNAHis accounts for a 200-fold range of expression. A variety of factors affect the level
and extent of tRNAHis charging, including anaerobic growth, low osmolarity, several antibiotics, and positive supercoiling. The
wealth of information concerning control of histidine synthesis results, in part, from the power of the available genetic systems.
Such genetic methods have permitted use of the his operon to study a variety of other basic phenomena, including gene
duplications and mutagenesis.
E. coli cannot use histidine as carbon or nitrogen source, although the related organism Klebsiella pneumoniae can. The pathway
in K. pneumoniae is the same for carbon or nitrogen source utilization. Histidine is degraded by a four-step pathway to glutamate,
ammonia, and formamide. Expression requires inactivation of a repressor by urocanate, the first intermediate in the pathway, and
activation by either cyclic-AMP and its binding protein or regulators of the Ntr response.

Further Reading
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, and Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene
knockout mutants: The Keio collection. Molecular Systems Biology 2: 2006.0008.
Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, and Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.
Nature Chemical Biology 5: 593–599.
Curtiss R (ed.) (2002) EcoSal-Escherichia coli and Salmonella: Cellular and Molecular Biology. In: Washington, DC: ASM Press. Modules 3.4.7, [Link], [Link], [Link], [Link],
[Link], [Link], and [Link], [Link]
Hu JC, Sherlock G, Siegele DA, Aleksander SA, Ball CA, Demeter J, Gouni S, Holland TA, Karp PD, Lewis JE, Liles NM, McIntosh BK, Mi H, Muruganujan A, Wymore F, and
Thomas PD (2013) PortEco: A resource for exploring bacterial biology through high-throughput data and analysis tools. Nucleic Acids Research 42: D677–D684.
Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BØ, and Agarwalla S (2006) Experimental and computational assessment of conditionally
essential genes in Escherichia coli. Journal of Bacteriology 188: 8259–8271.
Keseler IM, Mackie A, Peralta-Gil M, Santos-Zavaleta A, Gama-Castro S, Bonavides-Martı́nez C, Fulcher C, Huerta AM, Kothari A, Krummenacker M, Latendresse M, Muñiz-Rascado L,
Ong Q, Paley S, Schröder I, Shearer AG, Subhraveti P, Travers M, Weerasinghe D, Weiss V, Collado-Vides J, Gunsalus RP, Paulsen I, and Karp PD (2013) EcoCyc: Fusing model
organism databases with systems biology. Nucleic Acids Research 41: D605–D612.
Neidhardt FC (ed.) (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology. In: 2nd edn. Washington, DC: ASM Press. Chapters 3, 14, 18, 23–33, 36, 39, 41, 43,
44, 77, 92, and 94.
Reitzer L (2003) Nitrogen assimilation and global regulation in Escherichia coli. Annual Review of Microbiology 57: 155–176.
Schomburg I, Chang A, Placzek S, Söhngen C, Rother M, Lang M, Munaretto C, Ulas S, Stelzer M, Grote A, Scheer M, and Schomburg D (2013) BRENDA in 2013: Integrated
reactions, kinetic data, enzyme function data, improved disease classification: New options and contents in BRENDA. Nucleic Acids Research 41: D764–D772.
Yoshida M, Kashiwagi K, Shigemasa A, Taniguchi S, Yamamoto K, Makinoshima H, Ishihama A, and Igarashi K (2004) A unifying model for the role of polyamines in bacterial cell
growth, the polyamine modulon. Journal of Biological Chemistry 279: 46008–46013.
Yuan J, Doucette CD, Fowler WU, Feng XJ, Piazza M, Rabitz HA, Wingreen NS, and Rabinowitz JD (2009) Metabolomics-driven quantitative analysis of ammonia assimilation in
E. coli. Molecular Systems Biology 5: 302.

Relevant Websites
[Link] – BRENDA, the comprehensive enzyme information system.
[Link] – EcoCyc database of pathways, genes and gene regulation in Escherichia coli.
[Link]/main/ – Escherichia coli gene expression database.
[Link] – PortEco database of mutant phenotypes.