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Assembly of peptidoglycan

Assembly of peptidoglycan. Lengeler et al. Chapter 23 pp. 555-570 Assembly of cell structures 1. Functions of peptidoglycan 2. Peptidoglycan monomer 3. Three stages: enzymatic assembly 4. Similarity: penicillin versus D-Ala-D-Ala. Gram-negative cell envelope.

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Assembly of peptidoglycan

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  1. Assembly of peptidoglycan • Lengeler et al. • Chapter 23 pp. 555-570 • Assembly of cell structures • 1. Functions of peptidoglycan • 2. Peptidoglycan monomer • 3. Three stages: enzymatic assembly • 4. Similarity: penicillin versus D-Ala-D-Ala

  2. Gram-negative cell envelope

  3. 1. Functions of peptidoglycan • Structural integrity: sacculus • Cell shape • Cell division G+ and G- bacteria

  4. 2. Peptidoglycan monomer • 2 sugars: N-acetyl glucosamine (NAG) N-acetyl muramic acid (NAM) • Linkage = β 1  4 • NAG: unsubstituted • NAM: linked to 5 amino acids (aa) L-Ala, D-Glu, L-Lys (or DAP), D-Ala, D-Ala

  5. 3. 1st stage of assembly • 9 enzymatic reactions: cytoplasm • UDP-NAG --PEP + NADPH  UDP-NAM • Addition of each aa to UDP-NAM energy requiring rxns • L-Ala + ATP  UDP-NAM-L-Ala • D-Glu + ATP  UDP-NAM-L-Ala-D-Glu • DAP + ATP  UDP-NAM-L-Ala-D-Glu-DAP

  6. Peptidoglycan monomer • L-Ala –racemase D-Ala • D-Ala + D-Ala + ATP  D-Ala-D-Ala • D-Ala-D-Ala synthetase • both rxns inhibited by cycloserine • UDP-NAM-L-Ala-D-Glu-DAP + D-Ala-D-Ala  • UDP-NAM-L-Ala1-D-Glu2-DAP3-D-Ala4-D-Ala5

  7. 2nd stage of assembly • Transfer reactions: cytoplasmic membrane • C55 polyisoprenyl lipid~PP • synonym = bactoprenyl~P • Links to • UDP-NAM-L-Ala1-D-Glu2-DAP3-D-Ala4-D-Ala5 • NAM--(pentapeptide)-P-P-lipid

  8. Translocation across CM • -NAM—(pentapeptide)-P-P-lipid • + UDP-NAG •  NAG—NAM—(pentapeptide)-P-P-lipid • Inhibited by vancomycin, tunicamycin • Recycling of lipid • Inhibited by bacitracin • Adding monomers to pre-existing peptidoglycan **

  9. 3rd stage of assembly • a. chain formation • Adding more [NAG-NAM-pentapeptide] to existing urein sacculus • Transglycosylase: • NAG-NAM-NAG-NAM-P-P-lipid  • NAG-NAM-NAG-NAM-NAG-NAM-P-P-lipid • Each NAM is substituted with pentapeptide •  Uncrosslinked peptidoglycan

  10. 3rd stage of assembly • a. chain formation, cont’d • Uncrosslinked peptidoglycan  • Transpeptidase: • Cross linking of ε-NH2 group of DAP3 to…. • D-Ala4 on neighbouring strands •  cross linked peptidoglycan

  11. Bifunctional enzyme • Transglycosylase + Transpeptidase • = penicillin binding protein 1a [or 1b] • PBP 1a [or PBP1b] • Anchored in cytoplasmic membrane, facing periplasm

  12. 3rd stage of assembly • b. Maturation of peptidoglycan • Carboxypeptidase • Cleavage of D-Ala5 from cross linked peptidoglycan • -NAG-NAM-NAG-NAM-NAG-NAM-NAG- with each NAM substituted by tetrapeptide • PBP5, PBP6

  13. 4. Structural similarities • Penicillin versus D-Ala-D-Ala • Single target hypothesis • Inhibition of cross linking of peptidoglycan =transpeptidase • Multiple targets of penicillin action = PBPs

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