de novo synthesis

1. Pyrimidine Biosynthesis
Kuldeep Sharma
Devashish Somani
B.Tech Biotech.
Amity Institute of Biotechnology
Amity University Rajasthan

2. Introduction
• The biosynthesis of pyrimidine is a simpler process than the
purines.
• Pyrimidne synthesis is a de novo synthesis pathway involving
six step reactions.
• The six members pyrimidine ring is made first and then
attached to ribose-5-monophosphate.
• This pathway results in the synthesis of Uridine-5-
monophosphate (UMP).
• Out of 6 enzymes involved in this pathway, 5 are present in
the cytosol and one is present on the outer surface of inner
mitochondrial membrane.

3. Biosynthetic origins of pyrimidine ring atoms
Isotopic labeling experiments have shown that atoms N1,
C4, C5, and C6 of the pyrimidine ring are all derived from
aspartic acid, C2 arises from HCO3 , and N3 is contributed
by glutamine

4. Background History
The major breakthrough in the determination of the pathway for the de novo
biosynthesis of pyrimidine ribonucleotides was the observation that mutants of the
bread mold Neurospora crassa, which are unable to synthesize pyrimidines and
therefore require both cytosine and uracil in their growth medium, grow normally
when supplied instead with the pyrimidine orotic acid (uracil-6-carboxylic acid). This
observation led to the elucidation of the following six reaction pathway for the
biosynthesis of UMP

5. Metabolic pathway
STEP 1: Synthesis of carbamoyl phosphate
The first reaction of pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from
bicarbonate ion and the amide nitrogen of glutamine by the cytosolic enzyme carbamoyl
phosphate synthetase II (CPS II). This reaction is unusual in that it does not use biotin and
consumes two molecules of ATP: One provides a phosphate group and the other energizes the
reaction. We have previously discussed the synthesis of carbamoyl phosphate in connection
with the formation of arginine. The carbamoyl phosphate that is used to synthesize arginine via
the urea cycle is synthesized by a separate mitochondrial enzyme, carbamoyl phosphate
synthetase I (CPS I), which uses ammonia as its nitrogen source.

7. Metabolic pathway
STEP 2: Synthesis of carbamoyl aspartate.
Condensation of carbamoyl phosphate with aspartate to form carbamoyl aspartate is
catalyzed by aspartate transcarbamylase (ATCase). This reaction occurs without need
of ATP because carbamoyl phosphate is intrinsically activated.

9. Metabolic pathway
STEP 3: Ring closure to form dihydroorotate.
The third reaction of the pathway forms the pyrimidine
ring yielding dihydroorotate in an intramolecular
condensation catalyzed by the zinc metalloenzyme
dihydroorotase.

10. Metabolic pathway
STEP 4: Oxidation of dihydroorotate.
Dihydroorotate is irreversibly oxidized to orotate by dihydroorotate dehydrogenase
(DHODH). This eukaryotic enzyme, which contains FlavinMonoNucletide, is an integral
membrane protein that is located on the outer surface of the inner mitochondrial
membrane, where ubiquinone supplies its oxidizing power and gets converted to its
reduced form. The other five enzymes of pyrimidine nucleotide biosynthesis are
cytosolic in animal cells.

12. Metabolic pathway
STEP 5: Transfer of orotate to Ribose-5-monophosphate.
Orotate reacts with 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRPP) to yield
orotidine-5-monophosphate (OMP) in a reaction catalyzed by orotate
phosphoribosyltransferase and driven by hydrolysis of the eliminated PPi. This
reaction fixes the anomeric form of pyrimidine nucleotides in the configuration.
Figure: 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRPP)

14. Metabolic pathway
STEP 6: Decarboxylation to form UMP
The final reaction of the pathway is the decarboxylation of OMP by OMP
decarboxylase (ODCase) to form UMP.

17. Synthesis of Cytosine-tri-phosphate (CTP) from UMP
•Nucleoside monophosphate kinase (NMK) catalyzes transfer of Pi to
UMP to form UDP; nucleoside diphosphate kinase (NDK) catalyzes
transfer of Pi from ATP to UDP to form UTP
•CTP is formed by amination of UTP by CTP synthetase. In animals, the
amino group is donated by glutamine, whereas in bacteria it is supplied
directly by ammonia.

18. UMP UDP
ATP ADP ATP ADP
UTP
NMK NDK

19. Synthesis of Thymine Nucleotides
•Thymine nucleotides are made from dUMP, which derives
from dUDP, dCDP metabolism
•The enzyme Thymidylate synthetase converts dUMP to
dTMP through the methylation of dUMP .
•The methyl group is donated by N5,N10-methylene THF
•dUDPdUTPdUMPdTMP
dCDPdCMPdUMPdTMP

21. Regulation
In mammals, the pyrimidine biosynthesis is
regulated at two steps;
•Carbamoyl phosphate synthetase II, which is
inhibited by UDP and UTP and activated by
ATP and PRPP
•OMP decarboxylase, for which UMP and CMP
are competitive inhibitors.