CMB

Lecture on amino acid synthesis

D. Nelson, last modified Dec. 1, 2005

Reading: Berg, Tymoczko and Stryer 5th edition, Chapter 24

You are now familiar with the central metabolic pathways of glycolysis, gluconeogenesis 
and the TCA cycle. Now we will look at the metabolism of the amino acids. 
Before we start, I would like to introduce you to KEGG.

The Kyoto Encyclopedia of Genes and Genomes
The Kyoto Encyclopedia of Genes and Genomes index

The Kyoto Encyclopedia of Genes and Genomes or KEGG has maps of many metabolic 
pathways (134 metabolism maps).  They are building a resource that will allow easy comparison 
of pathways between genomes, and the assessment of an organism's biochemistry by 
analysis of its pathways.  The basic pathway maps have all known enzymes listed by their 
Enzyme Commission number (EC number).  Substrates and products are then placed in 
order with the enzymes that either make them or modify them.  The result is an automated 
generation of all possible pathways.  These can then be color coded for enzymes present in 
a single species.  Species maps are available by pull down menus. The newest twist to this method is to 
color code genes identified in gene chip arrays as being expressed in the same manner. 
These may be genes in a single  pathway or genes involved in the same process.
When whole genomes are analyzed, complete sets of pathways can be developed except for the 40-
50% of proteins that have no known function.  Gaps in pathways can be identified, and 
predictions made about an organism's metabolic strategies.  Essential pathways present in 
microorganisms but absent in humans are of course targets for antimicrobial drugs.  
Obvious examples are the vitamin biosynthetic pathways in bacteria.  Currently, the KEGG has 
analyzed >150 complete genomes including 32 eukaryotes, and they also include partial data on
21 draft eukaryotic genomes. 24 archaeal genomes and 260 bacterial genomes
are included.  You should try looking at some of the maps that are hyperlinked 
in the text below.  It may be interesting to compare yeast with E. coli and 
Methanococcus jannaschiito get a feeling for similarities and differences between 
the three main domains of life Archaea, Bacteria and Eukarya.

Clicking on an enzyme box will link you to the information on that gene, including the enzyme 
name the protein sequence and the nucleotide sequence and links to Locus Link and OMIM if available.  
If the Enzyme Commission has not named the enzyme yet, then it will have a partial number in the box.  
Related areas on other maps are also linked.  These are shown as circled regions with a label like urea 
cycle.  Clicking here will take you to the map that has that pathway.  

Mammals don't make all 20 amino acids, so it will be necessary to consider some of 
the pathways found in microorganisms.  In bacteria and other microorganisms, 
mutants that are defective for growth without a specific supplement are called 
auxotrophs.  Auxotrophs can be found in most biosynthetic pathways.

In going over these pathways, I will add information about some metabolic diseases.  
These have similar features.  Usually there is a build up of precursors in front of a block 
in a pathway. After the block, intermediates are absent or depleted. The total absence 
of an enzyme is not required to make people sick.  Low activity of an enzyme due to a 
point mutation or a premature stop codon can occur and this may lead to disease.  
Since genes in eukaryotes are built from exons, alternative splicing or exon 
skipping can cause disease.  We will see that there are many more diseases caused 
by defects in amino acid breakdown, than in amino acid biosynthesis.  That is due 
to accumulation of intermediates in the breakdown pathways that can act in 
deleterious ways. Many amino acids are neurotransmitters and high concentrations of 
them can lead to mental retardation.  Some intermediates are inhibitors of other 
enzymes and they can shut down needed metabolic activity.  These types of problems 
are not as likely in the biosynthetic pathways, because missing amino acids can be 
obtained in the diet, and feedback inhibition of pathways can shut down a pathway 
if intermediates accumulate to high levels.  The loss of a biosynthetic pathway can 
be harmful if an intermediate is required as a precursor in another pathway.  

Treatment of metabolic diseases is frequently dietary.  The accumulation of amino 
acids that are toxic can be controlled by a low protein diet supplemented with 
essential amino acids. Other possible treatments are transplants of cells, that can 
provide the missing enzyme. There have been several trials of treating Parkinson's 
disease (defect in L-dopa synthesis, in the tyrosine biosynthetic pathway) by 
surgical implants of fetal brain tissue into the brains of patients.  This has 
helped some patients.  There was one dramatic case of a man who had taken a street 
drug that was contaminated with a toxic chemical.  This resulted in killing the 
cells of his substantia nigra where dopamine is made and he became frozen, 
completely unable to move.  A transplant of fetal brain cells, done in Sweden, 
helped him to regain some movement.  

Not all defects in amino acid pathways result in disease.  Patients with 
hypermethioninemia are often normal, but they can have bad breath from 
dimethylsulfide production from the excess methionine.  

In biosynthesis, all 20 amino acids are made from only 7 precursors from the 
central metabolic pathways.  

1. fructose 6 phosphate precursor to aromatic amino acids
2. glyceraldehyde 3 phosphate precursor to aromatic amino acids
3. 3-phosphoglycerate precursor to 3-phosphogycerate family of amino acids
4. phosphoenolpyruvate precursor to aromatic amino acids
5. pyruvate precursor to pyruvate family of amino acids
6. alpha ketoglutarate precursor to alpha ketoglutarate family of amino acids
7. oxaloacetate precursor to aspartate family of amino acids

The Kyoto Encyclopedia of Genes and Genomes has 16 maps on amino acid metabolism.  
There is probably enough material for more than a dozen lectures, but 
we have only two, so you will be getting just the highlights of amino acid 
metabolism.  To give you an idea of what we are not going to cover, here is the map 
for phenylalanine metabolism.This does not include phenylalanine biosynthesis 
that is shown as a small circle near the top of the figure.

In plants and microorganisms, the 20 amino acids all receive their amino groups 
from glutamate in a transamination reaction.  In some pathways the transamination is the 
first step (methionine, threonine).  In other pathways, transamination is 
the last step (valine, leucine and isoleucine).  In mammals, only 10 amino acids 
are synthesized.  The rest are acquired in the diet.  

The source of nitrogen is clearly important.  The nitrogen in glutamate is 
incorporated from ammonia.  Ammonia is a reduced form of nitrogen that is created 
by reduction of N2 gas (nitrogen fixation) or by reduction of nitrate (nitrate 
assimilation).  Nitrogen fixation uses an enzyme called nitrogenase.  This enzyme 
contains molybdenum and complex iron-sulfur clusters.  Nitrogenase is poisoned by 
oxygen.  The N-N bond is very hard to break (225 kcal/mol).  It costs 16 ATP to reduce 
one nitrogen molecule to two ammonium ions.

Glutamate can be synthesized in two ways, by glutamate dehydrogenase in a reductive 
amination that couples ammonia to alpha ketoglutarate(p. 669), or by 
glutamate synthase that transfers the amide group from glutamine to alpha 
ketoglutarate (p. 670).  This last reaction makes two glutamates, one from 
glutamine and one from alpha ketoglutarate.  The glutamine used in this reaction 
is made by glutamine synthase. 

The two methods of forming glutamate are both present in bacteria.  However, glutamate 
dehydrogenase has a higher Km for ammonia than glutamate synthase.  In real life situations, 
where ammonia concentration is low, glutamate synthase is the preferred pathway.  Since 
glutamate synthase only transfers a nitrogen from glutamine, glutamine synthase is the key 
enzyme for introducing ammonia into carbon compounds. Glutamine synthase is a highly 
regulated enzyme in E. coli (see text, pp. 683-685). 

There are five families of amino acids.  Four are based on the precursors they are 
made from.  The fifth, the aromatic amino acids, all share the first part of their 
pathway up to chorismate.  Histidine is in a group all by itself. (fig. 24.7, p. 670)

Glutamate, glutamine, proline, arginine and lysine(in fungi, and lower eukaryotes) are all 
derived from alpha KG.  We have already seen how glutamate and glutamine are made.  

Proline is made starting from glutamate(see p. 673-674)  The gamma carboxyl group is 
activated by phosphorylation.  This compound is reduced and dephosphorylated to form  
glutamate gamma semialdehyde, a compound we will see again in amino acid degradation. 
The aldehyde spontaneously cyclizes by forming an internal Schiffs base with the alpha 
amino group.  This is then reduced to form proline.  

Hydroxyproline is made in collagen after the protein is synthesized.  The enzyme 
prolyl hydroxylase is an ascorbate dependent enzyme.  Lack of ascorbate causes 
scurvy.  Polar explorers traveling with Robert Scott to the South Pole in 1911/1912 
developed scurvy. This weakens collagen tissue.  One man who had suffered a bullet 
wound during a war began to have his wound reopen.  Ascorbate, or vitamin C is 
required to maintain scar tissue and to keep blood vessels from becoming fragile.  
None of Scott's polar expedition survived.  They traveled 1400 miles and died 
within 15 miles of their home base.  

Arginine is also made from glutamate via an activated gamma carboxyl group.  
However, the alpha amino group is acetylated first, so the gamma aldehyde cannot 
react with the alpha amino group to form a cyclic product .  After this is done, 
the aldehyde is produced and then glutamate donates the terminal amino group.  
The acetyl group is removed, leaving ornithine.  Ornithine is converted to arginine 
in the urea cycle. We will talk about the urea cycle when we cover amino acid degradation, 
so we will stop at ornithine for now.  N-acetylglutamate is an activator of an important enzyme, 
carbamoyl phosphate synthase I.  This enzyme catalyzes a major route of ammonia 
removal, so a defect in arginine biosynthesis leads to toxic levels of ammonia that 
result in death a few days after birth.  

Lysine can be made starting from alpha ketoglutarate in lower eukaryotes, but in 
higher eukaryotes, it is made from aspartate.  The fungal pathway of lysine 
synthesis is not shown in your book. This pathway has several steps similar 
to the TCA cycle.  Lysine has one more carbon than alpha ketoglutarate, so the 
starting compound must be lengthened. This is done by reacting acetyl CoA with 
alphaKG in a reaction that is similar to the citrate synthase reaction.  The 
product has an hydroxyl on the beta carbon, just like citrate.  This has to be 
moved to the alpha carbon in order to decarboxylate the homoisocitrate. Oxidation 
of the hydroxyl group to a carbonyl cannot proceed on a teriary alcohol.  It would 
make a pentavalent carbon.  The product alpha ketoadipate is similar to alpha 
ketoglutarate in the TCA cycle.  The compound is transaminated from glutamate to 
make a new amino acid alpha aminoadipate. It is an amino acid that is next in the 
series aspartate (4 carbons), glutamate (5 carbons), alpha aminoadipate (6 
carbons).  This compound is activated by phosphorylation and reduced to an aldehyde 
as was seen in proline and ornithine biosynthesis.  The aldehyde reacts with a 
glutamate amino group to form an adduct that is hydrolyzed on the other side of the 
nitrogen to form lysine and release alpha ketoglutarate. In purine 
biosynthesis aspartate is an amino donor like glutamate, that is cleaved to release 
fumarate.  The main difference here is the hydrolytic cleavage adds an oxygen back 
to the product.

The amino acids in this family include aspartate, asparagine, methionine, threonine, 
isoleucine and lysine by a different pathway from the one seen in fungi.  

Aspartate is formed by transamination of oxaloacetate from the TCA cycle.  Both alpha 
ketoglutarate and oxaloacetate are sources of carbon skeletons for amino acid synthesis.  
These amino acids are sources of nitrogen and carbon for building other amino acids and 
nucleotides.  These are exit points from the TCA cycle and these compounds have to be 
replenished or the cycle will run out of oxaloacetate to react with acetyl CoA.  (See 
p. 482)  The cycle is refilled by anaplerotic reactions that produce TCA cycle intermediates.  
The most important is pyruvate carboxylase that makes oxaloacetate from pyruvate and CO2.  
When there is a deficit of oxaloacetate, acetyl CoA builds up, because it cannot enter the 
TCA cycle.  Acetyl CoA activates pyruvate carboxylase.  Malic enzyme is another anaplerotic 
enzyme that converts pyruvate and CO2 to malate.  We will see later that all amino acids are 
broken down to TCA cycle intermediates, so amino acids can serve as sources of these 
intermediates to keep the TCA cycle going, even if carbohydrates are not available.  

Asparagine is made in two ways.  In bacteria, the amide nitrogen comes from ammonia, as 
in the glutamine synthase reaction.  In other organisms glutamine is the amino group 
donor to aspartate.  This reaction requires ATP hydrolysis to AMP and PPi.  (see p. 673)
The AMP group activates the carboxyl.  This is a recurring theme that you should be aware of.  

Threonine, methionine and lysine are all made starting from aspartate. 
Since it is important to make the appropriate amount 
of each amino acid, the first enzyme in the pathway, aspartokinase, is subject to 
regulation.  In fact in E. coli, there are three aspartokinase enzymes. (see p. 683)   
Aspartokinase I is feedback inhibited by threonine . Aspartokinase III is feedback inhibited by 
lysine.  Aspartokinase II is not feedback inhibited, but its gene expression is repressed by 
methionine.  Methionine is called a co-repressor(see p. 872) Lysine is a co-repressor of 
aspartokinase III and threonine and isoleucine (isoleucine is made from threonine) are 
co-repressors of aspartokinase I.  

The pathways for these 4 amino acids all start the same way.  Aspartate is phosphorylated 
and reduced to form the aldehyde, just as we saw with proline, ornithine and fungal lysine 
biosynthesis.   This requires an ATP and NADPH.  At this point lysine biosynthesis 
branches off, through an 8 step pathway that we won't cover in detail.  The main features 
are addition of three carbons as pyruvate to make a 7 carbon skeleton.  An extra amino 
group is added from glutamate and one carbon is removed in a decarboxylation reaction at 
the end of the pathway.  

The three remaining amino acids have one more common step before they branch off.  
This is the reduction of the aspartyl semialdehyde to an alcohol by NADPH.  The alcohol 
of aspartic acid is called homoserine.  This is very similar to threonine except the 
hydroxyl is on the gamma carbon instead of the beta carbon.  A two step pathway 
phosphorylates the gamma hydroxyl and shifts it to the beta carbon to make threonine.  

Homoserine is also the precursor for methionine.  Again the hydroxyl is activated, but 
this time it is by succinyl CoA that transfers a succinyl group and releases CoA.  
Methionine has a sulfur, and this is donated from cysteine.  The whole cysteine molecule 
is covalently attached to the activated carbon through the sulfur.  This is cleaved off as 
pyruvate and ammonia to leave the SH group on the product homocysteine.  The terminal 
methyl is donated by N5 methyl tetrahydrofolate.  

Methionine can react with ATP to form S-adenosylmethionine (SAM).  (see p. 676) 
The ATP is hydrolyzed to release PPi and Pi, so SAM 
costs three high energy phosphate bonds to make.  The methyl group on SAM is readily 
donated to methyl acceptors in synthetic reactions producing S-adenosyl homocysteine.  
This product is cleaved to form adenosine and homocysteine that are recycled.  Another 
route SAM can enter is the decarboxylation of the methionine part of the molecule to make 
decarboxylated SAM.  This structure is now a propylamine donor rather than a methyl donor.  
This group is transferred to a putrescine amino group (made by decarboxylation of 
ornithine) to make spermidine.  One more propylamine can be added to the other amino group 
of spermidine to make spermine.  

Isoleucine is made from threonine after it is oxidatively deaminated to form alpha 
ketobutyrate.  This compound is similar to pyruvate, except it has one more methylene 
group (p. 652). Pyruvate and alpha 
ketobutyrate are both processed by the same four enzymes to make valine and isoleucine.  
We will go over these in the pyruvate family of amino acids.  Since the deamination of 
threonine is unique to the isoleucine pathway, this step is feedback inhibited by 
isoleucine.  The next step is feedback inhibited by valine, so valine inhibits its own 
synthesis and isoleucine's as well, but isoleucine only inhibits its own pathway, not 
valine's.  (see pfig. 24.22, p. 682) (Remember isoleucine also inhibits aspartokinase I 
at the first step in its pathway.)

Alanine valine and leucine are the three members of this family.  Alanine is formed by 
transamination of pyruvate from glutamate.  Valine is made by the same pathway as 
isoleucine starting from the alpha keto acids pyruvate and alpha ketobutyrate.  In the 
first common step, pyruvate donates an hydroxyethyl group to the alpha carbon of the keto 
acid.  In the next step, the newly added acetyl group inserts itself between the alpha and 
beta carbons, forming a dihydroxy acid.  This is then dehydrated to make the alpha keto 
acids that are directly transaminated by glutamate to make valine and isoleucine.  

Leucine biosynthesis branches from alpha ketoisovalerate, the immediate precursor to 
valine.  This compound undergoes a reaction similar to oxaloacetate in the citrate 
synthase reaction.  It reacts with acetyl CoA to make a product that is both 
hydroxylated and carboxylated at the beta carbon.  As in the TCA cycle conversion of 
citrate, the hydroxyl is shifted to the alpha carbon and the carboxyl is removed as CO2.  
This forms a new alpha keto acid that is transaminated to form leucine.  

Serine, glycine and cysteine belong in this group.  3-phosphoglycerate is oxidized to 
convert the alpha hydroxyl to a keto group.  This is then transaminated and 
dephosphorylated to make serine.  (see p. 674) 

Glycine is made from serine in two different ways.  In the first, an hydroxymethyl group 
is transferred from serine to tetrahydrofolate to make N5 N10 methylenetetrahydrofolate.  
The second pathway is the glycine oxidase reaction acting in reverse.  Here CO2, NH4+ and 
N5 N10 methylenetetrahydrofolate condense to form glycine.  Serine is the source for one 
carbon units used in biosynthesis of many products like lipids, purines, thymine, 
tryptophan, cysteine and methionine.  Glycine is used in purine, glutathione and heme 
synthesis.  

There are several ways to make cysteine.  In bacteria, H2S can react with serine to make 
cysteine directly.  This requires a pyridoxal phosphate dependent enzyme.  In other 
organisms the hydroxyl is first activated by adding an acetyl group from acetyl CoA. The 
acetyl group is then displaced by H2S to form cysteine.  

Through this series of reactions, you should be noticing that there are common principles 
that are used in the pathways again and again.  Often, carboxyl groups are activated by 
phosphorylation and then reduced to make an aldehyde before some other reaction occurs.  
The series of reactions seen in the TCA cycle that react acetyl CoA with an alpha keto acid 
and then proceed to shift an hydroxyl group and decarboxylate the product, are used in the 
fungal lysine pathway and the leucine pathway.  There are a variety of activating groups 
that can be added to carboxyls and hydroxyls.  These include phosphates, CoA, succinate, 
acetyl groups or AMP.  You should be aware of similarities in the different pathways.  

The aromatic amino acids do not derive from a single metabolic intermediate starting point, 
like the other families of amino acids we already discussed.  It takes three intermediates 
from the glycolysis pathway to start aromatic amino acid biosynthesis.  Fructose-6-
phosphate and glyceraldehyde-3-phosphate are reconfigured by transketolase (see Fig. 20.21 
p. 566).  If you remember from the pentose phosphate 
pathway, transketolase moves a two carbon ketol group from a keto sugar to an aldose.  So 
the 6 carbon fructose-6-P becomes the four carbon erythrose-4-P and glyceraldehyde-3P picks 
up two carbons to become the 5 carbon xylulose-5-P (precursor to PRPP).  The erythrose-4-P 
reacts with phosphoenolpyruvate to start biosynthesis of the aromatic amino acids.

Phenylalanine, tyrosine and tryptophan are synthesized from the intermediate chorismate.  
This is also a precursor of many compounds in biology that contain a benzene ring.  
Vitamins E, K, folic acid, ubiquinone, plastoquinone and lignin are all made from 
Chorismate. Two carbohydrate skeletons phosphoenol pyruvate and erythrose 4 phosphate are condensed 
to form a seven carbon compound called 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP).  
(see fig. 24.16 p. 679)  This compound is cyclized with loss of the phosphate to form a six membered 
ring that undergoes dehydration and reduction to form shikimate.  This pathway is referred 
to as the shikimate pathway.  Shikimate is phosphorylated and then reacts with PEP, with the 
loss of phosphate from PEP to make the intermediate 3-enolpyruvyl-shikimate-5-phosphate.  
This loses its phosphate to become chorismate.  

Here the pathway branches in two directions to make trp on one branch and tyr and phe on 
the other branch.  

Tyrosine and phenylalanine are similar amino acids and their synthesis is similar(fig. 24.17 
p. 680).  The enolpyruvyl group on chorismate is shifted 
to the para position of the ring to make prephenate.  At this point the pathway diverges 
again.  If the carboxyl group is oxidized to CO2, the resulting alpha keto acid is 4-
hydroxyphenylpyruvate, which is transaminated from glutamate to form tyrosine.  If the 
carboxyl and the hydroxyl are both removed, the resulting alpha keto acid is phenylpyruvate.  
This is also transaminated to form phenylalanine.  

Tryptophan is also formed from chorismate, in a five enzyme pathway.  In the first step of 
this pathway, the enolpyruvyl group and the hydroxyl are removed and an amino group 
from glutamine is added to the ring, which is now aromatic.  This compound, anthranilate 
is condensed with PRPP, which you saw in purine biosynthesis.  The phosphoribosyl 
group is added to the amino group.  In step three, the ribose sugar is isomerized to a 
ribulose sugar.  The enzyme that catalyzes this step is one of the common genes in yeast 
that is used as a selectable marker TRP1.  Yeast that have a mutation in TRP1 cannot grow 
on media lacking tryptophan.  A functional copy of TRP1 on a plasmid complements this 
defect and can be selected for in a yeast transformation, on tryptophan minus plates.  If 
your favorite gene is on this plasmid, you can select for your gene at the same time, by 
selecting for trp plus colonies from a trp1 auxotrophic yeast.  

In step 4, the intermediate made by TRP1 is decarboxylated and dehydrated to form the 
indole ring of tryptophan, attached to glycerol three phosphate.  The last enzyme, 
tryptophan synthase actually has two active sites and two different steps are carried out on 
the alpha and beta subunits.  The glycerol three phosphate is removed as glyceraldehyde 3 
phosphate, on the alpha subunit and serine is added to replace it on the beta subunit.  The 
intermediate is transferred from the alpha to the beta subunit active sites through a tunnel 
on the enzyme.  This is called channeling.  (see figs. 24.19 and 24.20 p. 681).

The five enzymes in this pathway have been studied in many different organisms, including 
bacteria, fungi and yeast.  (see handout)  Some of the genes are fused together, so two 
enzymes are on one polypeptide chain.  Some of the enzymes have alpha and beta 
subunits, that are coded in separate proteins, but they have no activity unless they are 
associated with each other.  Anthranilate synthase is like this in all species, tryptophan 
synthase is like this in some species, in other species the alpha and beta subunit are fused 
as one protein.  Some of the proteins that are not sequential in the pathway are covalently 
linked by disulfide bonds.  All this points to the existence of a super complex of all the 
tryptophan biosynthetic enzymes.  However, this complex is pretty loose, since it cannot 
be isolated intact.  

In E. coli, there are three different DAHP synthase enzymes (first step in chorismate 
synthesis).  This is similar to aspartokinase in the thr, ile, met and lys pathways.  Each 
product, trp, tyr and phe inhibits one of the three different DAHP synthases.  The gene 
expression of these three enzymes is also repressed by the end products.  Trp represses the 
five enzymes unique to the trp pathway, since they are in a single operon.

In Bacillus subtilis, there is just one enzyme and it is not inhibited by the three amino 
acid products of the pathway.  Instead it is inhibited by chorismate and prephenate, the 
products that occur at the two branch points of the pathway.  The branches that lead to tyr 
and phe are inhibited by these amino acids, at the first step after prephenate is formed.  

The first step of histidine biosynthesis couples PRPP and ATP to make an unusual 
compound that has two ribose rings attached to the same adenine ring.  The compound 
has four phosphates (p. 697).  Two of these are cleaved off and then the adenine ring is broken.  
The ribose from PRPP is 
isomerized to the open chain conformation then, in reaction 5, glutamine donates its amide 
and displaces the remainder of the ATP molecule, leaving behind one nitrogen and one carbon 
from the adenine ring that was broken.  This cyclizes to make the imidazole ring.  Here we 
see imidazole coupled to glycerol phosphate.  This is like the tryptophan pathway, where the 
indole ring was coupled to glycerol phosphate.  Rather than swapping the glycerol phosphate 
for serine like we saw in the tryptophan pathway, histidine is made by dehydrating to make 
an alpha keto group (in yeast this enzyme is HIS3, a common auxotrophic marker) this 
compound is transaminated by glutamate.  The product is dephosphorylated and oxidized twice 
by the same enzyme histidinol dehydrogenase to make the carboxyl.  

The genes of the histidine biosynthetic pathway are in a single operon and they are 
repressed by histidine.

The histidine biosynthetic pathway is the basis for the Ames mutagenesis test.  Mutants in 
the histidine pathway are plated on a his minus medium and a test substance is included.  
The number of his+ colonies that appear compared to a control shows that a compound is 
mutagenic.  It changes the his minus mutants back to wild type and restores growth.  

Plants make all twenty amino acids, but humans make only 10.  Therefore, inhibitors of 
the pathways that are present in plants but not humans should not affect humans (and 
other mammals).  These are useful as herbicides.  (see p. 679)  The 
aromatic amino acids and histidine as well as the branched chain amino acids are all 
essential in humans, meaning we cannot make them.  (We can convert phenylalanine to tyrosine 
by a phenylalanine breakdown pathway, but we are missing the shikimate pathway.)  There are 
three inhibitors that block three of these pathways: the shikimate 
pathway, the histidine pathway, and an enzyme used in common to make Ile, Leu and Val.  
The popular garden weed killer Roundup (glyphosate) inhibits the shikimate pathway.

Recently, malaria has been shown to have an organelle called an apicoplast.  All members of the 
apicomplexan parasites have this plastid.  It appears to derive from a chloroplast like ancestor 
(a red alga) but it has lost the ability to make chlorophyll.  This makes malaria plant like. 
Recently several effors to identify drug targets unique to the apicoplast have identified 
candidates in lipid biosynthesis, a single pyruvate dehydrogenase gene, and a ferredoxin like 
a redox system needed for iron sulfur cluster synthesis.  The apicoplast 
has the 
shikimate pathway that should make it susceptible to roundup.