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Chapter ten

Biosynthesis of nucleotide
Topic: Synthesis of Pyrimidine and Purine nucleotide
Outline:
• Nucleic acid
• Nucleotide
• Nucleoside
• Nitrogen base
• Synthesis of Pyrimidine nucleotide
• Synthesis of Purine nucleotide
• Regulation Pyrimidine and Purine nucleotide synthesis.
Teaching and learning methods: Lectures, visual aid, interactive forms, assignment
and group discussion.
Reference books:
1.Bacterial Metabolism- Gerhard Gottschalk; 2nd Edition, chapter 3
At the end of this lecture student should be able to answer following questions.
• Define Nucleic acid, Nucleotide and Nucleoside.
• Mention the Nitrogen base for Purine and Pyrimidine nucleotide.
• Explain the synthesis of Pyrimidine nucleotide.
• Illustrate the synthesis of Purine nucleotide.
• Explain the regulation Pyrimidine and Purine nucleotide synthesis.
Nitrogenous Bases
• Planar, aromatic, and heterocyclic
• Derived from purine or pyrimidine
• Numbering of bases is “unprimed”
Nucleic Acid Bases
Purines Pyrimidines
Sugars
• Pentoses (5-C sugars)
• Numbering of sugars is “primed”

D-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group


Nucleic acid

• Nucleic acid a macromolecule consisting of


nucleotides; DNA and RNA are nucleic acids
Nucleoside
• A compound consisting of a purine or pyrimidine base and a pentose sugar.
• The nitrogenous bases of nucleotides include two types of purines and three
types of pyrimidines.
• A nucleotide consists of a nitrogenous base, a ribose or deoxyribose sugar, and
one or more phosphate groups.
• DNA contains adenine, guanine, cytosine, and thymine deoxyribonucleotides,
whereas RNA contains adenine, guanine, cytosine, and uracil ribonucleotides.
• The most common purines are adenine (A) and guanine (G), and the major
pyrimidines are cytosine (C), uracil (U), and thymine (T).
• The purines form bonds to a five-carbon sugar (a pentose) via their N9 atoms,
whereas pyrimidines do so through their N1 atoms
Nucleoside
• Purines bond to the C1’ carbon of the sugar at their N9 atoms
• Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms
Phosphate Groups
• Mono-, di- or triphosphates
• Phosphates can be bonded to either C3 or C5 atoms of the sugar.
Nucleotide
• A compound consisting of a purine or pyrimidine base, a five- carbon sugar, and a
phosphate.
• Result from linking one or more phosphates with a nucleoside onto the 5’ end of
the molecule through esterification
• The bases of nucleotides are planar, aromatic, heterocyclic molecules that are
structural derivatives of either purine or pyrimidine.
Nucleotide
• RNA (ribonucleic acid) is a polymer of ribonucleotides
• DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides
• Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine
—Ribonucleotides contain Uracil
—Deoxyribonucleotides contain Thymine
• Monomers for nucleic acid polymers
• Nucleoside Triphosphates are important energy carriers (ATP, GTP)
• Important components of coenzymes
—FAD, NAD+ and Coenzyme A
Synthesis of Pyrimidine nucleotide:
The precursors of the pyrimidine nucleotides are carbamoyl phosphate and
aspartate. The aspartate transcarboxylase condenses these compounds to yield
carbamoyl phosphate, which undergoes cyclization to give 4,5 dihydroortate.
Dehydrogenation then leads to orotate, the first intermediate containing the
pyrimiding ring. Ribose-5-P is activated by conversion into 5-phosphorybosyl-
pyrophosphate. 5-phosphorybosyl-pyrophosphate react with orotate and yield
orotidine monophosphate, which is subsequently decarboxylated to uridine
monophosphate (UMP). UMP can be converted into UTP by kinase reactions; a
subsequent amination reaction yields cytidine triphosphate (CTP). Amination
proceeds via a phophorybosyl intermediate.
Synthesis of Pyrimidine nucleotide:
• Cytosine
• Thymine
• Uracil

• The pyrimidine ring is synthesized as free pyrimidine & it is incorporated into


the nucleotide.
• Aspartate, glutamine (amide group) & CO2 contribute to atoms in the
formation of pyrimidine ring.
Step 1: Carbamoyl Phosphate Synthesis

• The reaction occurs in cytoplasm.


• Glutamine transfers its amido nitrogen to CO2 to produce carbamoyl
phosphate.
• This reaction is catalyzed by ATP dependent enzyme carbamoyl phosphate
synthetase II (CPS II).
• CPS-I is involved in urea cycle.
Step 2: Rate Limiting Step

• Carbamoyl phosphate condenses with aspartate to form carbamoyl


aspartate.
• This reaction is catalysed by aspartate transcarbamoylase.
• The atoms C2 & N3 are derived from carbamoyl phosphate & the rest are
from aspartate.
Step 3: Formation of Pyrimidine Ring
• Dihydroorotase catalyses the pyrimidine ring closure with a loss of H2O.
• Step 4: Oxidation
• NAD+ dependent dehydrogenation, leading to the formation of orotate.
• The enzyme is dihydro orotate dehydrogenase (DHODH).
Step 5: Formation of OMP
• Ribose 5-phosphate is now added to orotate to produce orotidine
monophosphate (OMP).
• This reaction is catalysed by orotate phosphoribosyltransferase.
• PRPP is the donor of ribose 5-phosphate.
Step 6: Decarboxylation
• OMP undergoes decarboxylation to uridine mono-phosphate (UMP).
• The enzyme is OMP decarboxylase (OMPDC).
• This is the first pyrimidine that is synthesized.
• 6-aza-uridine inhibits this step & used as an anticancer drug.
• Orotate phosphoribosyl transferase & OMP decarboxylase are domains of a
single protein.
• A defect in this bifunctional enzyme causes orotic aciduria.
Step 7: Synthesis of Triphosphates
• By an ATP-dependent UMP kinase reaction, UMP is converted to UDP which
serves as a precursor for the synthesis of dUMP, dTMP, UTP & CTP.
• UDP is phosphorylated to UTP (uridine triphosphate) with the help of ATP.
• The enzyme is nucleoside diphosphate kinase.
• Ribonucleotide reductase converts UDP to dUDP by a thioredoxin-dependent
reaction.
• Thymidylate synthetase catalyses the transfer of a methyl group from N5, N10 -
methylene tetrahydrofolate to produce deoxythymidine monophosphate
(dTMP).
Step 8: Formation of CTP

• Cytidine triphosphate (CTP) is synthesized from UTP by amination.


• CTP synthetase is the enzyme & glutamine provides the nitrogen.
• CTP synthetase needs ATP.
UMP
ATP
UMP Kinase
ADP
Thioredoxin (2SH)
UDP Nucleoside
Thioredoxin (-S-S-) diphosphate Kinase
Ribonucleotide
reductase ATP
dUDP ADP UTP
Glutamine + ATP + H2O
dUMP CTP
N5 , N10 synthetase
Methylene
THF Glutamate + ADP + Pi

THF CTP
dTMP
Regulation of Pyrimidine synthesis
• In bacteria, aspartate transcarbamoylase (ATCase) catalyses a committed step in
pyrimidine biosynthesis.
• ATCase is a good example of an enzyme controlled by feedback mechanism by
the end product CTP.
• Carbamoyl phosphate synthetase II (CPS II) is the regulatory enzyme of
pyrimidine synthesis in animals.
• It is activated by PRPP and ATP & inhibited by UDP & UTP.
• OMP decarboxylase, inhibited by UMP & CMP, also controls pyrimidine
formation.
Biosynthesis of Purine Ribonucleotides
Formation of purine ring
• N1 of purine is derived from amino group of aspartate.
• C2 & C8 from formate of N10 - formyl THF.
• N3 & N9 are obtained from amide group of glutamine.
• C4, C5 & N7 are contributed by glycine.
• C6 directly comes from CO2.
• The purines are built upon a pre-existing
ribose 5-phosphate.
• Liver is the major site for purine nucleotide
synthesis.
• Erythrocytes, polymorphonuclear leukocytes
& brain cannot produce purines.
Steps in Purine Biosynthesis
• Ribose 5-phosphate, of carbohydrate metabolism is the starting material for
purine nucleotide synthesis.
• It reacts with ATP to form phosphoribosyl pyrophosphate (PRPP).
• Glutamine transfers its amide nitrogen to PRPP to replace pyrophosphate &
produce 5-phosphoribosylamine.
• PRPP glutamyl amidotransferase is controlled by feedback inhibition of
nucleotides (IMP, AMP & GMP).
• This reaction is the 'committed.
• Phosphoribosylamine reacts with glycine in the presence of ATP to form
glycinamide ribosyl 5-phosphate or glycinamide ribotide (GAR).
• Catalyzed by synthetase.
• N10-Formyl tetrahydrofolate donates the formyl group & the product formed is
formyl glycinamide ribosyl 5-phosphate.
• The reaction is catalyzed by formyl transferase.
• Glutamine transfers the second amido amino group to produce formyl
glycinamidine ribosyl 5-phosphate.
• The reaction is catalyzed by synthetase.
• The imidazole ring of the purine is closed in an ATP dependent reaction to yield 5-
aminoimidazole ribosyl 5-phosphate.
• The reaction is catalyzed by synthetase.
• Incorporation of CO2 (carboxylation) occurs to yield aminoimidazole carboxylate
ribosyl 5-phosphate.
• The reaction is catalyzed by carboxylase.
• Does not require the vitamin biotin or ATP.
• Aspartate condenses with the aminoimidazole carboxylate ribosyl 5-phosphate to
form aminoimidazole 4-succinylcarboxamide ribosyl 5-phosphate.
• The reaction is catalyzed by synthetase.
• Adenosuccinate lyase cleaves off fumarate & only the amino group of aspartate is
retained to yield aminoimidazole 4-carboxamide ribosyl 5-phosphate.
• N10-Formyl tetrahydrofolate donates a one-carbon moiety to produce 5-
formaminoimidazole 4-carboxamide ribosyl 5-phosphate.
• Catalyzed by formyltransferase.
• The final reaction catalyzed by cyclohydrolase leads to ring closure with an
elimination of water molecule.
• The product obtained is inosine monophosphate (IMP), the parent purine
nucleotide from which other purine nucleotides can be synthesized.
Synthesis of AMP & GMP from IMP
Synthesis of AMP:
• Ionosine monophosphate (IMP) is the immediate precursor for the formation of
AMP & GMP.
• Aspartate condenses with IMP in the presence of GTP to produce
adenylsuccinate which, on cleavage, forms AMP.
Synthesis of GMP:
• IMP undergoes NAD+ dependent dehydrogenation to form xanthosine
monophosphate (XMP).
• Glutamine then transfers amide nitrogen to xanthosine monophosphate (XMP) to
produce GMP.
• 6-Mercaptopurine is an inhibitor of the synthesis of AMP & GMP.
• It acts on the enzyme adenylsuccinase (of AMP pathway).
• IMP dehydrogenase (of GMP pathway).
Synthesis of AMP & GMP

Aspartate + GTP
IMP NAD+
Adenylsuccinate
synthetase IMP Dehydrogenase
GDP + Pi NADH + H+

Adenosylsuccinate Xanthosine monophosphate


Gluatamine + ATP + H2O

Adenylsuccinase GMP Synthetase


Formate
Gluatamate + AMP + Pi

AMP GMP
6-Mercaptopurine
Formation of di & tri-phosphates
• The nucleoside monophosphates (AMP & GMP) converted to the corresponding
di & triphosphates.
• This is achieved by the transfer of phosphate group from ATP, catalysed by
nucleoside monophosphate (NMP) kinases & nucleoside diphosphate (NDP)
kinases.
Formation of di & tri-phosphates

Nucleoside monophosphate (AMP, GMP)


ATP
NMP kinase
ADP

Nucleoside diphosphate (ADP, GDP)


ATP
NDP kinase
ADP

Nucleoside triphosphate (ATP, GTP)


Inhibitors of purine synthesis
• Folic acid (THF) is essential for the synthesis of purine nucleotides.
• Sulfonamides are the structural analogs of para-aminobenzoic acid (PABA).
• These sulfa drugs can inhibit the synthesis of folic acid by microorganisms.
• This indirectly reduces the synthesis of purines & nucleic acids (DNA & RNA).
• The structural analogs of folic acid (e.g. methotrexate), used to control cancer.
• They inhibit the synthesis of purine nucleotides & nucleic acids.
• These inhibitors also affect the proliferation of normally growing cells.
• Azaserine (diazo acetyl-L-Serine) is a glutamine antagonist & inhibits reactions
involving glutamine.
• Other synthetic nucleotide analogues used as anticancer agents are 6-thio
guanine & 8-aza guanine.
Regulation of purine nucleotide synthesis
• The intracellular concentration of PRPP regulates purine synthesis.
• This is dependent on the availability of ribose 5-phosphate & the PRPP
synthetase.
• PRPP glutamyl amidotransferase is controlled by a feedback mechanism by purine
nucleotides.
• If AMP & GMP are available in adequate amounts, their synthesis is turned off at
the amidotransferase reaction.
• Another important stage of regulation is in the conversion of IMP to AMP & GMP.
• AMP inhibits adenylsuccinate synthetase while GMP inhibits IMP dehydrogenase.
• AMP & GMP control their respective synthesis from IMP by a feedback
mechanism.
Regulation of Purine Biosynthesis, Mammals
Purine regulation demonstrates a couple of different strategies reflecting different
growth and evolutionary strategies

E. coli
Regulation in E. coli is more complex, reflecting the primacy of this synthesis in
growth and replication:
Conversion of ribonucleotides to deoxyribonucleotides

• The synthesis of purine & pyrimidine deoxyribonucleotides occurs


from ribonucleotides by a reduction at the C2 of ribose moiety.
• This reaction is catalyzed by a multisubunit (two B1 & two B2
subunits) enzyme, ribonucleotide reductase.
Formation of deoxyribonucleotides from
ribonucleotides

Ribonucleotide reductase
Ribonucleoside Ribonucleoside
diphosphate (ADP, diphosphate (ADP,
GDP,CDP, UDP) GDP,CDP, UDP)

Thioredoxin (2SH-Reduced) Thioredoxin (S-S-Oxidized)

Thioredoxin Reductase

NADP+ NADPH + H+
• Supply of reducing equivalents:
• The enzyme ribonucleotide reductase itself provides the hydrogen atoms needed
for reduction from its sulfhydryl groups.
• The reducing equivalents, in turn, are supplied by thioredoxin, a monomeric
protein with two cysteine residues.
• NADPH-dependent thioredoxin reductase converts the oxidized thioredoxin to
reduced form which can be recycled again & again.
• Thioredoxin thus serves as a protein cofactor in an enzymatic reaction.
Regulation of deoxyribonucleotide synthesis:
• Deoxyribonucleotides are mostly required for the synthesis of DNA.
• The enzyme ribonucleotide reductase maintains the adequate supply of
deoxyribonucleotides.
• Ribonucleotide reductase is a complex enzyme with multiple sites (active site &
allosteric sites) that control the formation of deoxyribonucleotides.
Synthesis of purine nucleotide