Download Chapter 22 Biosynthesis of amino acids, nucleotides and related

Chapter 22
Biosynthesis of amino acids,
nucleotides and related
molecules
1. Reduction (fixation) of N2 into ammonia (NH3 or NH4+)
2. Synthesis of the 20 amino acids.
3. Synthesis of other biomolecules from amino acids
4. The de novo pathways for purine and pyrimidine
biosynthesis.
5. The salvage pathways for purine and pyrimidine reuse.
1. The nitrogenase complex in certain
bacteria (diazotrophs,固氮生物)
catalyzes the conversion of N2 to NH3
• The nitrogen in amino acids, purines, pyrimidines and other
•
•
•
•
biomolecules ultimately comes from atmospheric nitrogen.
Cyanobacteria (蓝藻细菌, photosynthetic) and rhizobia (根
瘤菌, symbiont) can fix N2 into NH3.
The reduction of N2 to NH3 is thermodynamically favorable :
N2 + 6e- + 6H+
2NH3 G`o = -33.5kJ/mol
But kinetically unfavorable: the bond energy for the triple
bond in N2 is 942 kJ/mol.
• The nitrogenase (固氮酶) complex mainly consists
of two types of enzymes: the dinitrogenase and the
dinitrogenase reductase.
• The dinitrogenase (containing molybdenum，钼,
thus called the MoFe protein) is a tetramer of two
different subunits, containing multiple 4Fe-4S
centers and two Mo-Fe clusters.
• The dinitrogenase reductase (also called the Fe
protein) is a dimer of two identifcal subunits,
containing a single 4Fe-4S redox center.
• The nitrogenase complex is highly conserved among
different diazotrophs.
Cyanobacteria and Rhizobia can fix N2
into ammonia
Rhizobia exist in
nodules of
leguminous plants
The nitrogenase complex
The dinitrogenase
reductase (dimer)
The dinitrogenase
(tetramer)
The dinitrogenase
reductase (dimer)
3Fe-3S
Fe-Mo cofactor
3Fe-3S
Mo
ADP 4Fe-4S
4Fe-4S
ADP
4Fe-4S
(P-cluster)
2. Electrons are transferred through
a series of carriers to N2 for its
reduction on the nitrogenase complex
• Eight electrons are believed to be needed for
each round of fixation reaction: with six for
reducing one N2 and two for reducing 2 H+
(to form H2).
• The electrons mainly come from reduced
ferredoxin (from photophosphorylation) or
reduced flavodoxin (from oxidative
phosphorylation) and are transferred to
dinitrogenase via dinitrogenase reductase.
• For each electron to be transferred from
dinitrogenase reductase to dinitrogenase, two ATPs
are hydrolyzed causing a conformational change
which reduces the electron affinity for the reductase
(i.e., an increased reducing power).
• The oxidized and reduced dinitrogenase reductase
dissociates from and associates with the
dinitrogenase, respectively.
• The nitrogenase is in imperfect enzyme: H2 is
formed along with NH3.
• The overall reaction catalyzed is:
• N2 + 8H+ +8e- + 16ATP + 16H2O 
•
2NH3 + H2 + 16ADP + 16Pi
Electrons are transferred
to N2 bound in the
active site of dinitrogenase
via ferredoxin/flavodoxin
and dinitrogenase reductase
3Fe-3S
3Fe-3S
N2 is believed to bind to
the cavity of the Fe-Mo
cofactor of the
dinitrogenase active site.
3. The nitrogenase complex is
extremely labile to O2 and various
protective mechanisms have evolved
• Some diazotrophs exist only anaerobically.
• Some cyanobacterial cells develop thick walls
to prevent O2 from entering.
• The bacteria in root nodules are isolated from
O2 by being bathed in a solution of the
oxygen-binding protein leghemoglobin.
Leghemoglobin, produced in legume plants,
has a high affinity to O2 and protects the
nitrogenase complex in rhizobia
4. Reduced nitrogen in the form of
+
NH4 is assimilated into amino acids
via a two-enzyme pathway
• First NH4+ is added to the side chain of glutamate to
form glutamine in an ATP-dependent reaction
catalyzed by glutamine synthetase.
• Then the side chain amino group of Gln is further
transferred to a-ketoglutarate to form Glu in a
reaction catalyzed by glutamate synthase, an
enzyme only present in bacteria and plants, not in
animals.
• The amide group of Gln is a source of
nitrogen in the synthesis of a variety of
compounds, such as carbamoyl phosphate,
Trp, His, glucosamine-6-P, CTP, and AMP.
• The amino groups of most other amino acids
are derived from glutamate via
transamination.
Newly fixed nitrogen
in the form of NH4+ is
first incorporated into
glutamate to form
glutamine
The side chain
amino group of
glutamine is then
e
transferred to
a-ketoglutarate
to form Glu
5. The bacterial Glutamine
synthetase is a central control point
in nitrogen metabolism
• The bacterial glutamine synthetase has
12 identical subunits (each having an
independent active site) arranged as two
hexagonal rings.
• Each subunit of the enzyme is
accumulatively inhibited by at least eight
allosteric effectors.
• In addition the enzyme is more susceptible to the
allosteric inhibition by having Tyr397 residue
modified by adenylylation.
• The addition and removal of the AMP group to
the glutamine synthetase are catalyzed by two
active sites of the same bifunctional
adenylyltransferase (AT).
• The substrate specificity of AT was found to be
controlled by a regulatory protein, PII.
• The activity of PII, in turn, is regulated by the
uridylylation of a specific Tyr residue: PII-UMP
stimulates the adenylylation activity of AT, however,
the unmodified PII stimulates the deadenylylation
activity of AT.
• The addition and removal of UMP to PII, in turn, are
again catalyzed by two active sites of the same
protein, uridylyltransferase (UT): a-ketoglutarate
and ATP stimulate the uridylylation, however, Gln
and Pi stimulate the deuridylylation (thus
adenylylation of AT, inactivating glutamine
synthetase).
The bacterial glutamine synthetase
has 12 subunits arranged as two
rings of hexamers
Active
sites
Tyr397
(adenylylation site)
The glutamine
synthetase is
accumulatively
inhibited by at least 8
allosteric effectors,
mostly end products
of glutamine
metabolism
A specific Tyr residue in bacterial
glutamine synthetase can be reversibly
adenylylated
6. Amidotransferases catalyze the
transfer of the amide amino group
from Gln to other compounds
• All of the enzymes contain two structural domains:
one binding Gln, the other binding the amino group
accepting substrate.
• A Cys residue is believed to act as a nucleophile to
cleave the amide bond, forming a covalent glutamylenzyme intermediate with the NH3 produced remain
in the active site and react with the second substrate
to form an aminated product.
A proposed
action
mechanism for
amidotransferases
7. The 20 amino acids are synthesized
from intermediates of glycolysis, the
citric acid cycle, or pentose phosphate
pathway
• The nitrogen is provided by Glu and Gln.
• As in amino acid degradation, some of the synthetic
pathways (e.g., for Ala, Glu, and Asp) are very
simple, but others (e.g., the aromatic ones) are very
complex.
• Most bacteria and plants can synthesize all 20, but
mammals can only synthesize about 10, with 10
being “essential”, needed in the diet.
• The pathways for the biosynthesis of the 20
amino acids can be categorized into six
families according to the metabolic
precursors used.
+ ATP
Gln
Intermediates of glycolysis,
the citric acid cycle, and
pentose phosphate pathway
serve as precursors for
synthesizing the 20 standard
amino acids.
+ acetyl-CoA
+ pyruvate
+Asp
8. a-ketoglutarate is the common
precursor for the biosynthesis of Glu,
Gln, Pro, and Arg in bacteria
• To make Glu, the amino group can be from the side
chain of Gln (catalyzed by an amidotransferase,
glutamate synthase in bacteria) or ammonia
(catalyzed by glutamate dehydrogenase in
vertebrates).
• Gln is made from Glu by obtaining an amino group
from ammonia in a reaction catalyzed by glutamine
synthetase.
• Proline is synthesized from Glu via one step of
phosphorylation (activation), two steps of enzymecatalyzed reductions (using NADPH) and one step
of nonenzymatic cyclization reaction (forming a
Schiff base).
• To make Arg, ornithine is first made from Glu with a
few steps similar to that of Pro synthesis, except that
an acetyl group is first added to the a-amino group
of glutamate to protect the spontaneous Schiff base
formation between the aldehyde group and the
amino group and removed after an amino group is
transferred to the aldehyde group.
• Arg is then synthesized from ornithine using steps
similar to those of urea cycle in bacteria.
• Arg from the diet can be converted to Pro in
mammals by being converted to ornithine first using
the urea cycle enzymes and then to 1-pyrroline-5carboxylate by ornithine d-aminotransferase.
• Similarly, Arg can be formed from glutamate gsemialdehyde (an intermediate of Pro synthesis) also
by using ornithine d-aminotransferase.
Glu is synthesized from a-ketoglutarate
by an amination reaction with amino
groups obtained from Gln or ammonia
Gln is synthesized from Glu
by obtaining an amino group
from ammonia in a reaction
catalyzed by glutamine
synthetase
Glutamine
synthetase
Glutamamate
kinase
Acetylglutamate
synthase
Glutamate
dehydrogenase
spontaneous
Pyrroline carboxylate
reductase
aminotransferase
9. Ser, Gly, and Cys are all derived
from 3-phosphoglycerate
• Ser is synthesized (in all organisms) from 3phosphoglycerate via NAD+-dependent oxidation,
PLP-dependent transamination, and
dephosphorylation reactions.
• Gly is either derived from Ser by removal of the side
chain b-carbon (catalyzed by serine
hydroxymethyltransferase, using PLP and H4 folate)
or synthesized (in vertebrate liver cells) from CO2
and NH4+ (catalyzed by glycine synthase, using
N5,N10-methylene H4 folate).
• In plants and bacteria, Cys is made from Ser by
replacing the side chain –OH group (after being
activated first by acetylation) by an –SH group using
sulfide (硫化物) reduced from sulfate (硫酸盐).
• In mammals, Cys is made from Met (providing the
sulfur atom after being converted to homocysteine)
and Ser (providing the carbon skeleton).
Ser is synthesized from 3-phosphoglycerate via oxidation,
transamination, and dephosphorylation reactions
Phosphoserine
aminotransferase
Gly is either derived from Ser by removal of the side chain b-carbon
or synthesized (in vertebrate liver cells) from CO2 and NH4+
Serine
hydroxymethyl
transferase
Glycine
Synthase
Cys is derived from Ser
via a two-step pathway
using sulfide in plants
and bacteria
Cys is synthesized
from homocysteine
(which is derived
from Met) and Ser
in mammals
Homocysteine (高半胱氨酸）
(胱硫醚b-合成酶)
(胱硫醚g-裂解酶)
Homocysteine is derived from Met via S-adenosylmethionine and Sadenosylhomocysteine intermediates
Methionine
adenosyl
transferase
Met
ATP
Methionine
synthase
Methyl
transferase
Hydrolase
Homocysteine
10. Ala, Val, and Leu are derived
from pyruvate
• Ala is synthesized from pyruvate via a simple
transamination reaction (with amino group donated
by Glu).
• Val is synthesized from the transferring of a twocarbon unit to a pyruvate, forming a-acetolactate,
which is then converted via isomerization,
reduction, dehydration, and transamination.
• The immediate precursor of Val, a-ketoisovalerate, is converted to Leu via four steps
of reactions: the addition of an acetyl group,
a position switching of an –OH group, an
oxidative decarboxylation, and a
transamination reaction.
Val is synthesized
from two pyruvates
Dihydroxy acid
dehydratase
Acetohydroxy
acid synthase
Valine
aminotransferase
Acetohydroxyl acid
isomeroreductase
Val
Leu is synthesized from a-keto-isovalerate
Dehydrogenase
Synthase
Aminotransferase
Isomerase
Leu
11. Asp, Asn, Thr, Met are all derived
from oxaloacetate
• Asp is synthesized from oxaloacetate via a simple
transamination reaction.
• Asn is synthesized from Asp by an amidation (酰胺
化) reaction with Gln donating the NH4+(catalyzed
by an amidotransferase).
• Thr and Met are all derived from Asp, branching off
after Asp is converted to aspartate b-semialdehyde
(in a way similar to Glu to glutamate gsemialdehyde conversion).
• Thr is synthesized from aspartate bsemialdehyde via homoserine.
• Met is also synthesized from homoserine: it
is first activated via succinylation; the
accepting the sulfur atom from cysteine and
methyl group from N5-methyl H4 folate.
Asp is synthesized
from oxalocaetate by a
simple transamination
Transaminase
Asn is synthesized
from Asp by transferring
an amino group from
the amide group of Gln
Asparagine
synthetase
Thr is synthesized from
Asp via homoserine
Threonine
synthase
Homoserine
kinase
Homoserine
Asp is first
dehydrogenase
converted to
aspartate-b-semialdehyde which is then used
to synthesize Thr, Met, and Lys.
Met is also synthesized from
homoserine via cystathionine
and homocysteine
Homoserine
acyltransferase
Cystathionineg-synthase
Cystathionineb-lyase
Methionine
syntase
Met
12. Lys and Ile are derived from
oxaloacetate and pyruvate
• Lys is synthesized from aspartate b-semialdehyde
(which is derived from Asp) and pyruvate
(contributing to the two carbons at positions of d and
e of Lys), with glutamate donating the e-amino
group.
• Ile is made from a-ketobutyrate (the
dehydration/deamination product of Thr) and
pyruvate, using the same set of enzymes as that for
Val synthesis.
Lys is synthesized from
aspartate-b-semialdehyde
and pyruvate
Dihydropicolinate
synthase
Dihydropicolinate
synthase
Dehydrogenase
Obtaining the e-amino
group from Glu
Synthase
Aminotransferase
Removing the carboxyl group
of the condensed pyruvate
Desuccinylase
Epimerase
Decarboxylase
Lys
Pyruvate
Thr
Acetolactate
synthase
Acetolactate
syntase
Ile is made from Thr and
Pyruvate using the same set
Of enzymes for Val synthesis
Acetohydroxy acid
isomeroreductase
Acetohydroxy acid
isomeroreductase
a-isopropylmalate
synthase
Isopropylmalate
isomerase
Dihydroxyl acid
dehydratase
Dehydrogenase
Valine
aminotransferase
Leucine
aminotransferase
Ile
Val
Leu
13. The aromatic amino acids are
derived from phophoenoylpyruvate
and erythrose 4-phosphate
• All aromatic amino acids share the first seven
reactions for biosynthesis, up to the formation of
chorismate (分支酸).
• All the carbons of the aromatic ring are derived from
PEP and erythrose 4-P: ring closure follows the
condensation reaction; which in turn is followed by
step-wise double bond introduction (by dehydration).
• A second PEP enters to make chorismate.
• Chorismate can be converted to anthranilate (by
accepting an amino group from Gln and releasing a
pyruvate) or prephenate (by switching the position
of the PEP component via a Claisen rearrangement)
by the catalysis of anthranilate synthase or
chorismate mutase respectively.
• Anthranilate (邻氨基苯甲酸 ) can be further
converted to Trp via another four steps of reaction:
accepting two carbons from 5-phosphoribosyl-1pyrophosphate (PRPP), and three carbons from Ser.
• Prephenate (预苯酸) can be converted to Phe
by releasing the carboxyl and hydroxyl groups
from the ring, and accepting an amino group
from Glu via a transamination reaction.
• Prephenate can also be converted to Tyr by an
oxidative decarboxylation and a
transamination reaction.
The biosynthesis of all
aromatic amino acids
share the first seven
steps of reactions
four steps
three steps
one step
two steps
one step
two steps
four steps
Chorismate, formed from
the condensed product of
PEP and erythrose 4-P, is
the common precursor of
Trp, Tyr and Phe.
The 2nd PEP
enter here
Ring formation
step
2nd double
bond
1st double
bond
Chorismate
Isomerase
Synthase
Synthase
(邻氨基苯甲酸 )
Transferase
Trp
Synthase
Chorismate is converted to Trp by
accepting an amino group from Gln, two carbons from 5 phosphoribosyl1-pyrophosphate (PRPP), and three carbons from Ser.
Chorismate
mutase
Dehydrogenase
Claison
rearrangement
dehydratase
Chorismate can also be
converted to prephenate
which serve as the
common precursor of
Phe and Tyr biosynthesis
14. Tryptophan synthase catalyzes
the conversion of indole-3-glycerol
phosphate to Trp with indole as an
intermediate
• The tetrameric enzyme has two a and two b
subunits.
• Indole-3-glycerol phosphate enters the a subunits
(the “lyase”) and is cleaved to form indole and
glyceraldehyde-3-P.
• Serine enters the b subunits, forms a Schiff base
with PLP, and is dehydrated to form a PLPaminoacrylate adduct.
• The indole intermediate formed on the a subunit
then enters the active site of the b subunit via a
“substrate channel”, where it condenses with the
PLP-aminoacylate intermediate to form a ketimine
(酮亚胺) intermediate, which is then converted to
a aldimine (醛亚胺) intermediate.
• The aldimine intermediate is then hydrolyzed to
form Trp.
The a and b subunits of tryptophan
synthase have different enzymatic
activities with substrate channeling
A ketimine
An aldimine
The hydrophobic indole is channeled from the a to
the b subunits in tryptophan synthase
15. Histidine is derived from three
precursors: 5-phosphoribosyl 1pyrophosphate, ATP and Gln
• The synthesis begins with the condensation of ATP
and PRPP: N-1of the purine ring is linked to the C-1
of the ribose.
• For the final synthesis of Histidine, PRPP
contributes five carbons, ATP contributes one
nitrogen and one carbon (both from the purine ring),
Gln contributes one nitrogen, and Glu contributes
the 3rd nitrogen (of the a-amino group).
• The unusual synthesis of histidine from a
purine is taken as evidence supporting the
hypothesis that life originated from RNA:
the synthetic pathway is considered as a
“fossil” of the transition from RNA to the
more efficient protein-based life forms.
Transferase
Hydrolase
The synthesis of histidine begins with the
condensation of PRPP and ATP: The C-1 of
the ribose ring is linked to the N-1 of the purine
ring.
A purine!
Hydrolase
Cleaved between
C-2 and N-3
Amidotransferase
Ring opens between
N-1 and C-6
Isomerase
Aldose
Ketose
Dehydratase
Histidine is made from PRPP,
ATP, Gln and Glu.
Aminotransferase
Dehydrogenase
16. Several feedback inhibition
mechanisms are found to work in
regulating amino acid biosynthesis
• The first case of allosteric feedback inhibition
was discovered in studying Ile biosynthesis in
E. coli
• The enzyme catalyzing the first reaction
(threonine dehydratase) is inhibited by the end
product (Ile) in a synthetic pathway.
• Several intricate feedback inhibition
mechansims have been found in branched
pathways for amino acid biosyntheses.
• In enzyme multiplicity, several isozymes are present
to catalyze a common step of reaction: each isozyme
responds to a different allosteric modulator, avoiding
the inhibition of a common reaction by only one end
product.
• In concerted inhibition, one enzyme is inhibited by
two or more than two modulators, with effect more
than additive.
• In sequential feedback inhibition, the end products
inhibit enzymes catalyzing the branching points only,
while the initial committing step is inhibited by
common precursors of the end products.
The first case of
allosteric feedback
inhibition was
discovered in
studying Ile
synthesis in E. coli
In branching pathways leading to
the synthesis of multiple end
products from common precursors,
several forms of feedback inhibition
mechanisms are used to coordinately
regulate the synthesis of all the
amino acids.
Several intricate feedback inhibition mechansims have been found in
branched pathways for amino acid biosyntheses
Sequential
Enzyme multiplicity
Concerted
High level of one (e.g., Y)
should not prevent the synthesis
of the other (e.g., Z).