Molecular Microbiology Chapter 2

·      Two N-acetylhexosamines: N-acetylglucosamine (Glc-NAc) and N-acetylmuramic acid (Mur-NAc)

·      Aminoacids: L-alanine, D-alanine, D-glutamic acid and a di-aminated amino acid (L-Lysin for Gram-Positive and meso-diaminopimelic acid for Gramm negative)

• Synthesis through polymerization of disaccharide peptide units (M and G) linked over an betha 1-4 bound and form the glycan chain.
• Each M saccharide has e chain with the peptide (L-Ala-D-Glu-Meso-Dap (L-Lys)-D-Ala-D-Ala) (the D-Glu iscoupled over its gamma and nod alpha carboxyl group)
• Two side bridges are linked together and the energy needed for this comes from de D-Ala-D-Ala hydrolysis, the linking varies form species, for E-colie the Carboxyl group from D-Ala (4) binds to the NH₂ group from the diaminated residue (3)

• Three major steps occur in the three different places, cytoplasm, membrane and periplasm
• First step(plasma): UDP-Glc-Nac and UPD-Mue-NAc-pentapetide
  • UPD-Glc-Nac is a common metabolite
  • UPD-Mue-NAc-pentapetide has to be made from UPD-Glc-Nac by the six enzymes MurABCDEF
    • A: PEP ist attached and then the phosphate group removed
    • B: DB (where phosphate was removed) is reduced
    • C: L-Alanine is added
    • D: D-Glu is added
    • E: diaminated residue is added
    • F: D-ala D-ala is added

• Second step (plasma membrane):
  • Membraneintegral lipid carrier binds to the Mur-NAc-pentapeptide (lipid 1) then Glc-NAc is added (lipid 2)
  • MurJ flipase flips over the disaccharide-pentapeptide across the membrane
• Third step (Periplasm):
  • Transglycosylase (TGs) brings and binds the disaccharide-pentapeptide to a growing peptidoglycan chain
  • Trasnpeptidation reaction cross-links a “donor” D-Ala (4) with the amine of the meso-DAP(acceptor) via two steps:
    • 1. Donor acyl-D-Ala-D-ala forms an Acyl-Enzyme complex and on D-Ala (5) is released
    • 2- Amino group from acceptor binds to it and the enzyme is released
    • Carboxypeptidase controls the process

• Peptidoglycan is in almost all bacteria and only found theirà ideal target for antimicrobial chemotherapy
• Different compounds attacking different targets of the peptidoglycan assembly have been developed

• Largest family of antibiotics used, several hundreds of β lactam have been synthesized
• 4 main families, differ in activity, sensitivity to β lactamase

• β-lactams antibiotics function as structural analogs of the acyl-D-Ala-D-Ala but its reaction is irreversibly
• Transpeptidase reacts irreversibly with the β-lactam antibiotics and produce a stable penicilloyl-enzyme complex which cannot be hydrolyzed with water (Water cannot penetrated into the active site)

• Using ³H-Penicillin they can be visualized
• Β-Lactam are also bacteriocidal, not only stop bacterial growth but also kill them
• The cell wall is weakened an no longer to able to hold the pressure
• the peptidoglycan is under constant remodeling (for cell growth), Model proposes that a multi-enzyme complex goes along the peptidoglycan structure and replaces one strand with tree new strands

• β lactamase destroys the lactam ring of those antibiotic -->  no antimicrobial activity

• are evolutionary related to the transpeptidase and have a bit larger active site, allowing water to hydrolys the penicilloyl-enzyme complex
• β lactamase have lost their original transpeptidase activity

• β lactamase help to protect enzymes outside the cell à in Gramm-positive thy are secreted in negative they are localized in the periplasm (more cos-efficient, no losses by diffusion)

• Their expression in carefully controlled
• Inducer is a muropeptide (is a product from PG from uncontrolled hydrolysis from β-lactam inhibition, this mechanism differs from classical systems where the substate (e.g Lactose) induces the sysnthesis of the enzyme involved in its breakdown. This regulation makes sense since the targets of β lactam are in the periplasm and transcription machinery in the cell, if β lactam would regulate it, then the reaction would take to long and the bacteria would dye before

• Cyclic gylcopeptide active against Gramm-positive (cannot diffuse across the OM of -)
• Inhibits as well the PG synthesis but complexes terminal D-Ala-D-Ala from lipid 2 àtranspeptidase is not possible
• Coupling the drug to fluorescent dye it was possible to study, where and how peptidoglycan assembles
• Last active frontline drug against some staphyslococcy that have resistance against penicillins and other antibioticas à causes problems in hospitals
• Enterococci replaced Acyl-D-Ala-D-Ala by Acly-D-Ala-D-latcice acid, this modification only needed two enzymes:¨à resistant to vancomycin
  • VanH pyruvate, produces D-Lactate
  • VanA ligase, couples D-lactate to D-Ala
• High-level resistance uses VanX, which stops the normal pathway
• Terminal group of the pentapeptide does not play any role in the crosslinking à function of crosslinking is still normal strength
• Vancomycin is 1000 lower binding affinity to the Lac
• The Van… enzymes are encoded by the van… genes

• Structural analog of D-alanine and inhibits alanine racemas (L-Ala into D-Ala) and also inhibits D-Ala-ligase, which dimerizes D-Ala

• Peptide antibiotics like bacitracin interfere with the C55-cycle
• C55 cycle shuttels the disaccharide-pentapeptide from the cytoplasm into the periplasm