This document summarizes the process of peptidoglycan biosynthesis in bacterial cells and mechanisms of several antibiotics that target this process. It discusses the three main stages of peptidoglycan synthesis: monomer synthesis in the cytoplasm, translocation and polymerization at the inner membrane surface, and polymer cross-linking. Key enzymes and intermediates involved in each stage are identified. Several classes of antibiotics that inhibit peptidoglycan synthesis are then described, including their mechanisms of action and mechanisms of resistance.

1. *
SARATH T M
AMRITA
UNIVERSITY

2. *Since peptidoglycan essential for bacterial survival
*Its biosynthesis is the major target of most antibiotics
*The largest and widely used bacterial cell wall synthesis
inhibitors is the β lactam antibiotics
*It inhibits transpeptidases enzyme, which inhibits peptide cross
linking.

3. *
*Peptidoglycan biosynthesis occurs in three main steps
1. Monomer synthesis
2. Glycan polymerization
3. Polymer cross linking
*The first stage is intracellular and involves murein monomer synthesis
from amino acids and sugars
*The second and third stage involves the export of murein monomers to
the surface of inner membrane, followed by their polymerization into
linear peptidoglycan polymers and their cross linking into two
dimensional lattices and three dimensional mats

4. Glucose
amidation
+phosryln
Glucosamine-1-P
acetylCoA UDP
GlmU
UDP-NAG
PEP
MurA MurB
Phosphomycin
UDP-NAM
A DE K/DAP
MurEMurC MurD
UDP-NAM
DE
A
K
UDP-NAM
A
DE
K
DA
DA
A A
Alanineracemase
D-A-D-Asynthetase
MurF
Cycloserine
STAGE-1: MUREIN MONOMER SYNTHESIS
cytoplasm
MurE
MurD
4

5. 5
*The “murein monomer "is a disaccharide comprising
Nacetylglucosamine connected via a beta linkage to the C4 hydroxyl
of N-acetyl muramic acid
*The first phase of peptidoglycan synthesis takes place in the
cytoplasm and involves the conversion of UDP-N-acetyl glucosamine
(UDP-NAG), to UDP-N-acetyl muramic acid.
*MurA , also known as enolpyruvate transferase, transfers
enolpyruvate from phospho enolpyruvate (PEP) to UDPNAG to
form UDP-NAG pyruvate enol ether.
*The flavoenzyme MurB (also known as UDP-NAG enolpyruvate
reductase) reduces the double bond to produce UDP-NAM

6. 6
*MurC , MurD , and MurE sequentially add the amino acids L-
alanine, D-glutamate, and a diamino acid—either L-lysine or
diaminopimelic acid (DAP) —to UDP-NAM.
* DAP differs from lysine in having a carboxyl group as well as an
amine on the side chain.
* Most Gram- positive bacteria use L-lysine, whereas a minority of
Gram-positive and all known Gram-negative bacteria use DAP.
*DAP is not found in humans, therefore it offers a unique target for
antibiotics.

7. 7
*Peptide formation continues with the addition of D- alanyl- D- alanine
dipeptide (D- ala-D- ala) to the growing chain.
*The dipeptide is synthesized from two molecules of L- alanine in two
reactions.
*The first reaction requires the transformation of L-alanine to D- alanine.
*This reaction is catalyzed by the enzyme alanine racemase.
*In the second reaction, an ATP- dependent enzyme called D-ala-D-ala
synthetase joins the two alanines together.
*The resulting D- ala-D- ala dipeptide is added to the UDP- NAM
tripeptide by MurF

8. Cell memb
Periplasmic
space
cytoplasm
BP
UDP-NAM
A
DE
K
DA
DA
UMP
BP-NAM
A
DE
K
DA
DA
MraY
UDP-NAG
UDP
BP-NAM-NAG
A
DE
K
DA
DA
BP-NAM-NAG BP
A
DE
K
DA
DA
G 5
tRNA
MurG
NAM-NAG
A
DE
K
DA
DA
G 5
NAM-NAG – NAM-NAG
A
DE
K
DA
DA
G 5
A
DE
K
DA
DA
G
5
vancomycin
Transglycosylase
bacitracin
STAGE-2: MUREIN MONOMER TRANSLOCATION & POLYMERIZATION
8

9. 9
*The second phase of peptidoglycan synthesis takes place on the inner
surface of cytoplasmic membrane and begins with the transfer of
UDP- NAM peptide to a phospholipid carrier embedded in the
membrane.
*This carrier is called Bactoprenyl phosphate, because murein
monomers are assembled on it, delivered by it to the surface of
plasma membrane, and then released in a process that regenerates the
carrier for further cycles of reaction
*The reaction by which UDP- NAM peptide is anchored to this carrier
lipid is mediated by an integral protein called MraY.
* Once the NAM peptide is anchored to the carrier lipid, a membrane
associated enzyme called MurG catalyzes the transfer of NAG to the
C4 hydroxyl of the NAM sugar to produce a lipid anchored NAM-
NAG disaccharide commonly known as LIPID II
*Polymerization is catalyzed by enzymes called Peptidoglycan
glcosyltransferases (PGTs)

10. NAM-NAG
A
DE
K
DA
DA
G G G G G
NAM-NAG
A
DE
K
DA
DA
G G G G G
NAM-NAG
A
DE
K
DA
DA
G G G G G
NAM-NAG
A
DE
K
DA
G G G G G
transpeptidase
ß-lactams
STAGE-3: POLYMER CROSS LINKING 10

11. 11
*In the final stage of cell wall synthesis, murein chains are cross linked to
one another by enzymes called transpeptidases (TPs).
*TPs were first identified as the molecular targets of Penicillin, so they
are also called Penicillin- binding proteins.
*The PGT domain couples murein monomers to produce glycan strands.
*These oligosaccharide chains must then be cross linked to produce the
murein found in bacterial cell wall.

12. 12
*
PHOSPHOMYCIN AND PHOSMIDOMYCIN
*Inhibit murein monomer synthesis by inhibiting the synthesis of UDP-
NAM from UDP- NAG.
*Phosphomycin is a PEP analogue that inhibits bacterial enolpyruvate
transferase by covalent modification of enzyme’s active site.
*PEP is a key intermediate in mammalian glycolysis, but these agents
does not interfere with carbohydrate metabolism in human cells.
*This selectivity of antibacterial action is likely caused by structural
differences between the mammalian and bacterial enzymes that act on
PEP.
*PHOSMIDOMYCIN another PEP analogue acts by the same
mechanism as Phosphomycin.

13. 13

*Cycloserine, a structural analogue of D-ala, is a second line agent used to
treat multi drug resistant M. tuberculosis infection.
*Cycloserine inhibits both alanine racemase that converts L- Ala to D- Ala
and the D-Ala-D-Ala ligase that joins together two D-Ala molecules.
*Cycloserine is an irreversible inhibitor of these enzymes and binds to
them more tightly than the natural substrate D-Ala.
*Resistance of Cycloserine includes over expression of alanine racemase
and mutations in alanine uptake system.

14. 14

*It is a peptide antibiotic that interferes with the dephosphorylation of
Bactoprenyl diphosphate.
*It is notable among the anti cell wall agents for having a lipid as target.
*Bacitracin inhibits dephosphorylation by forming a complex with
bactoprenyl diphosphate that involves bacitracin’s imidazole and
thiazoline rings.
*Due to its significant kidney, neurological and bone marrow toxicity,
bacitracin is not used systemically.
*It is most commonly used topically for superficial dermal or
ophthalmologic infections.

15. 15
*
VANCOMYCIN
*Vancomycins are glcopeptides with bactericidal activity against Gram-
positive rods and cocci.
*These agents interrupt cell wall synthesis by binding tightly to the D-
Ala-D-Ala terminus of the murein monomer unit, inhibiting
peptidoglycan polymerization and thereby blocking addition of murein
units to the growing polymer chains.
*Toxicity of vancomycin causes this agents to be used only when an
infection is found to be resistant to other agents.
*Its toxicity includes skin flushing or rash
*Vancomycin has also been associated with nephrotoxicity and
ototoxicity, particularly when other ototoxic or nephrotoxic medications
such as gentamycin are co administered.

16. 16
*
β- lactam Antibiotics (Penicillin, Cephalosporin, Monobactams,
Crabapenems)
*Largest and most widely prescribed class of antibiotics that inhibits
bacterial cell wall synthesis.
*The different agents in this class vary in chemical structure and
consequently in spectrum of action.
*But all β- lactams share the same antibiotic mechanism of action:
inhibition of murein polymer cross linking
*Chemically, the key to the mechanism of action is the presence of a four
membered β lactam ring.
*This ring makes every β lactam a structural analogue of terminal D-Ala-
D-Ala dipeptide.
*B-lactams have affinity for several or specific transpeptidases-

17. 17
*
Ethambutol, Pyrazinamide and Isoniazid
*Used widely in treating tuberculosis
*Ethambutol, a bacteriostatic agent, decreases arabinogalactan synthesis
by inhibiting the arabinosyl transferase that adds arabinose units to the
growing arabinogalactan chain.
*Pyrazinamide and Isoniazid inhibit mycolic acid synthesis.
*Pyrazinamide is a pro drug; it must be converted to its active form,
pyrazinoic acid, by the enzyme pyrazinamidase.
*Pyrazinoic acid inhibits FAS1, the enzyme that synthesizes the fatty
acid precursors of mycolic acid.
*Isoniazid targets FAS2 complex.

18. 18
RESISTANCE----
ETHAMBUTOL
*Mutation/over expression of target enzymes
ISONIAZID
*Mutation in catalase- peroxidase which is needed for the activation of
the drug

19. 19
*
*primarily used for resistant gram negative infections.
*Polymyxin B binds to the cell membrane and alter its structure
making it more permeable.
*They are cationic polypeptides which make electrostatic &
hydrophobic interactions with anionic components like phospholipids
& lipopolysaccharide
*Little to no effect on gram-positive since cell wall is too thick to
permit access to membrane.

20. 20
*

21. 21
*
RIFAMPIN
* Semisynthetic derivative of naturally occurring antibiotic Rifamycin B
* Exerts bactericidal activity against mycobacteria by forming a highly stable complex with
the DNA- dependent RNA polymerase, thereby inhibiting RNA synthesis.
* The drug targets β subunit of bacterial RNA polymerase
* Rifampin permits the initiation of transcription, but then blocks elongation once the length
of nascent RNA reaches 2 to 3 nucleotides.
* Used in combination with Isoniazid
* Another example is Streptolydigins

22. 22
*
AMINOGLYCOSIDES (Streptomycin)
*Used mainly in treating infections caused by Gram negative bacteria
*Aminoglycosides bind to 16s rRNA of 30s subunit and elicit
concentration- dependent effects on protein synthesis.
*At low concentrations, aminoglycosides induce ribosomes to misread
mRNA during elongation, leading to synthesis of proteins containing
incorrect amino acids.
*Aminoglycosides interfere with mRNA decoding function of 30s
subunit.

23. 23
*RESISTANCE----
*Plasmid encoded transferase enzyme which
inactivates the drug
*Alteration or elimination of porins or other proteins
needed for drug transport
*Mutation of target site

24. 24
*
*Tetracyclines binds reversibly to the 16s rRNA of the 30s subunit and
inhibits protein synthesis by blocking the binding of aminoacyl tRNA
to the A site on the mRNA- ribosome complex.
*This action prevents the addition of further amino acids to nascent
peptide.
RESISTANCE---
*Plasmid encoded drug efflux pumps
*Production of proteins that interfere with the binding of tetracycline
to ribosome
*Enzymatic inactivation of drug

25. 25
*
*Binds to 23SrRNA & inhibits peptide bond formation (interferes with
the peptidyl transferase activity of 23srRNA)
*Because of serious toxicities used only if safer alternatives are not
available
*Used occasionally for typhoid, bacterial meningitis , rickettsial
disease etc.
RESISTANCE----
*Plasmid encoded acetyl transferases inactivate the drug

26. 26
*
*Binds to 23S rRNA of 50s subunit
*Block the exit tunnel from which the nascent peptide emerge ( block
translocation step)
*Important in treating pulmonary diseases (e.g.: Legionnaire’s disease)
RESISTANCE---
*Mutation in target site
*Increased drug efflux activity
*Methylase production by gram positives (modifies ribosomal target)

27. INHIBITORS OF NUCLEOTIDE SYNTHESIS
27

28. Steps in nucleotide synthesis
Synthesis of ribonucleotides
Reduction of ribonucleotides to deoxyribonucleotides
Conversion of dUMP to dTMP
28

29. PURINES
PYRAMI
DINES
base ribonucleoside ribonucleotide
deoxyribonucleo
side
deoxyribonucleo
tide
adenine adenosine AMP deoxyadenosine dAMP
guanine guanosine GMP deoxyguanisine dGMP
cytosine cytidine CMP deoxycytidine dCMP
uracil uridine UMP deoxyuridine dUMP
thymine deoxythymidine dTMP- -
reduction
NUCLEOTIDES
29

30. Target the DNA synthesis ( S-phase of cell cycle)
Applied in both infectious & neoplastic diseases
30

31. PURINE SYNTHESIS PYRAMIDINE SYNTHESIS
Aminoacids PRPP
IMP
Folate
Folate
precursors
AMP GMP
amination oxidation
dAMP dGMP
(reduction)
UMP CMP
amination
dUMP
dTMP dCMP
reduction
reduction
methylation
NUCLEOTIDE SYNTHESIS
Aspartate
Orotate
Carbomyl
phosphate
PRPP
31

32. In the de novo purine synthesis pathway, two separate steps utilize 10-formyl-THF.
32

33. Folate-requiring reactions, collectively referred to as one-carbon
metabolism, include those involved in phases of amino acid
metabolism, purine and pyrimidine synthesis, and the formation
of the primary methylating agent, S-adenosylmethionine (SAM )
methyltransferase
reactions
10-formyl-THF
33

34. INHIBITORS OF FOLATE METABOLISM
BACTERIA
BACTERIA &
HUMANS
Pteridine+ PABA
Dihydropteroic acid
Pteridine+ PABA
Dihydropteroic acid
dihydrofolate
Dihydropteroate
synthase
sulfonamides
glutamate
tetrahydrofolate
DHFR
dTMP
5 FU
Flucytosine
Trimethoprim
Methotrexate
Pyrimethamine
TS
34

35. SULFA DRUGS
• Sulfonamides & sulfones
• PABA analogues
• Inhibits dihydropteroate synthase &prevents folic acid synthesis
in bacteria
• Bacteriostatic
• Highly selective drugs---mammalian cells do not express
dihydropteroate synthase
35

36. RESISTANCE---
• Overproduction of endogenous PABA (70 fold more than normal)
• Mutation in target enzyme
• Decreased permeability of bacterial membranes to sulfonamides
• Sulfones –used in leprosy treatment (Dapsone)
36

37. METHOTREXATE ( MTX)
• DHFR inhibitor
• Folate analogue– close structural resemblance with natural
substrate
• Used in cancer chemotherapy
• Rapidly growing cancer cells have increased need of purines
& thymidylate
• Malignant cells more susceptible to apoptosis inducing effects
of MTX
37

38. methotrexate
pyrimethamine
trimethoprim
38

39. TRIMETHOPRIM
Folate analogue
Inhibits bacterial DHFR
PYRIMETHAMINE
Folate analogue
Inhibits parasitic DHFR
Only effective drug against toxoplasmosis
Most common drug for malaria also
39

40. 5-FLUROURACIL (5-FU)
 possibility1
• 5-FU is converted into FdUMP (5-fluro-2’-deoxyuridylate)
• which in turn inhibit TS
• Cells undergo thymine less death
Possibility2
• 5-FU can be metabolized to FUTP( flox uridine triphosphate)
• FUTP can be incorporated into mRNA in place of UTP
• Interferes with mRNA processing
40

41. • Toxic effect of 5-FU can be either single/combinational
• TS inhibition is considered as the dominant mechanism
• Mostly used as anti neoplastic drug
FLUCYTOSINE
• Highly selective anti fungal drug
41

42. pm
Flucytosine
Cytosine deaminase
5-FU
5-FdUMP
dUMP dTMP
Thymidylate synthase
MECHANISM OF ACTION OF FLUCYTOSINE
42

43. IMP Adenylo
succinate
AMP
ATP
dAMP
dATP
DNA
Ribonucleotide
reductase
Hydroxyurea
6-Mercaptopurine
XMP
IMPDH
GMP
dGMP
dGTP
DNA
Ribonucleotide
reductase
INHIBITORS OF PURINE NUCLEOTIDE SYNTHESIS
ASS
43

44. 6- MERCAPTOPURINE (6-MP)
• Inosine analogue (contains S at C-6)
• Inhibits inosine monophosphate dehydrogenase & adenylo
succinate synthase
• Major application in acute lymphoblastic leukemia
HYDROXYUREA
• Inhibits ribonucleotide reductase by scavenging a tyrosyl
radical from the active site of the enzyme
44

45. UMP UTP CTP
dCTP
Ribonucleotide
reductase
DNA
Hydroxyurea
dUMP
Ribonucleotide
reductase
dTMP
dTTP
DNA
INHIBITORS OF PYRAMIDINE
SYNTHESIS45