Bio 406 biosynth peptidoglycan

What are the guidelines for macromolecule biosynth

formed from monomers 2 monomers are activated, usually involving binding to carrier molecule 3directional couplingdue to chem differences in polymer chain ends, 4monomer added in condensation RX with → H2Oas a byproduct 5 carriers are recycled

The 4 enzyme general characteristics

1enzymes are usually proteins

2 enhance the rate of a reaction by lowering the activation energy

3catalysts are not permanently changed or consumed

4bring substrate or reactants together in the environment suitable for rx

What are the 7 steps of the peptidoglycan subunit

1 convert F6P to NAG requires GIn, acetyl CoA and binding to UTP/UDP

2 convert NAG bound carrier to NAM posphoenolpyruvate

3 sequential addition of alternating L and D amino acids

4 transfer to membrane bound carrier

5 addition of NAG

6 modification of pentapeptide if required

7 translocation across cytoplasmic membrane and added to the peptidoglycan chain

What 8 enzymes are involved in peptidoglycan biosynt

1 transferases

2 racemases (convert L to D)

3 autolysins modify peptidoglycan strands to add additional subunits to allow for division and growth of bacteria

4 transglycosylases and class A PBPs (penicillin binding proteins )=ß1-4glycosiclic bond

5 transpeptidases perform the cross linking between d-alanine #4 and NH2 AA #3 or penta glycine (requires both class A and B PBPs

6 carboxypeptidase removes terminal d-alanine = delta trade for new peptide bond

7 endopeptidase modifies strands for growth

8 phosphatases removes a phosphate

How does cyanide regulate the enzymes

Binds to the iron group of cytochrome c oxidase and shuts down oxidative phosphorylation

What does penicillin do to regulate enzymes

Reversible binding of carboxypeptidase transpeptidase binding

How does vancomycin inhibit enzymes

Binds d-ala - d-ala terminus inhibiting transfer across the membrane

Autolysins

Relatively stable enzymes that partially digest peptidoglycan, tightly regulated at transcription level

Carrier molecules

UDP cytoplasmic carrier and energy source, Bactroprenel lipid carrier, phosphorylated (tagged to the cytoplasmic membrane) binds to NAm pentapeptide/UDP at release of UMP (recycles to UTP)

What process occurs in the cytoplasm

Initial biosynthesis (synthesis of nag and nam, addition of pentapeptide)

What process occurs at the cytoplasmic membrane/cytoplasmic side

Bound to cytoplasmic membrane carrier add nag to nam decorate the pentapeptide if required

What occurs in the perplasmic space

Flipped across the membrane and growing strand occurs

What are the 4 steps of the synthesis of soluble precursors:UDP-NAM (forming NAM from NAG)

1. UMP phosphorylation to UTP

2. Condensation of UTP+NAG-1P yielding UDP-NAG

3. Transfer of enol group from PEP yielding UDP-NAG-Pyruvate enol ether

4. Reduction by NADPH yielding UDP-NAM

What does phophonomycin do?

Binds to the enol transferase and blocks the formation of UDP-NAG-pyruvate enol ether

What are the 3 steps of the synthesis of soluble precursors: UDP-NAM pentapeptide (formation of the pentapeptide)

1. Sequential addition of alternating L and D amino acids (Requires ATP and either Mg2+)

2. L forms converted to D forms via racemases

3. D-Ala-D-Ala added as a dipeptide

   1. Third amino acid in the chain must be a do-amino amino acid so that the free NH2 can be used for cross linking

Different genera my have different compositions of pentapeptide what are the last two peptides of Erysipelothrix and Enterococcus

D-ala-Dlactose, D-Ala-D-Ser

What are the 3 steps of the transfer of precursors to membrane bound carrier lipids?

1. Bactroprenol lipid carrier, phosphyrlated binds to NAM and pentapeptide-UDP and releases UMP

2. Addition of UDP-NAG with loss of UDP

   1. now that you have the complete peptide subunit the modification of the pentapeptide via Glycyl-tRNA can occur if needed.

What are the steps of disaccharide pentapeptide translocation across the cytoplasmic membrane (2)

1. Transfer of the disaccharide/pentapeptide subunit to the growing peptidoglycan strand with formation of beta1-4 (C1 on NAG and C4 on NAM) glycosidic bond

2. energy via transglycosylation when bactoprenol dissociates (it is diphosphorylated)

What are the two steps of crosslinking via transpeptidation

1. Transpeptidase penicillin binding protein class a and b perform the crosslinking between D-alanine #4 and amino terminal of amino acid #3 or penta glycine

2. Carboxypeptidase remove the terminal D-alanine producing energy that can be traded for a new peptide bond

Penicillin, cephalosporins carbapenems

Have a B-lactate ring that looks like a binding site on D-Ala-D-Ala and thus bind to the enzyme transpeptidase (irreversible) reversible binding to D-Ala carboxypeptidase

Vancomycin and Ristocetin

High molecular weight binds irreversibly to D-Ala-D-Ala and stops transfer across the membrane Ristocetin not used as an antibiotic as it causes platelet aggregation

Bacitracin

Binds to diphosphate bactoprenol and blocks phosphates thus inhibiting carrier recycling

Cycloserine

CNS toxicity (used only for TB if you have a very resistant form) Competitive inhibitor for D-Ala-D-ala Stops crosslinking by binding the enzyme.

Phosphomycin

Binds to the enol transferase halting that transfer

Lantibiotics

Form pores in the bacterial lipid bilayers and complex with bactoprenol resulting in accumulation of cytoplasmic intermediates eventually triggering autolysis

Carbapenems

B-lactate antibiotics originally drug of last resort to treat extended spectrum B-lactamase producing Gram negative bacteria which can inactivate both penicillins and cephalosporins

Carbapenem-resistant Enterobacteriaceae (CRE)

Two variant mechanisms carbapenemases producing and other or non-carbapenemases altered portions and or pumps

More serious carbapenemases are frequently carried on mobile genetic elements and thus can be transmitted from bacterial species to bacterial species via transduction, transformation or conjugation

What are the risk factors of CRE infection in the GI tract

1. Extended use of health care facilities for unrelated comorbidities

2. Extended use of varied antibiotics

3. Immune compromised state

4. Mechanical ventilation and in dwelling medical devices

5. Travel to an endemic area

What is HEPT

Heterozygous phage therapies:

synthetic enzymes that bind peptidoglycan or endopeptidase domains or other target.

They impact cytosolic membrane integrity, 16S rRNAse activity or other nuclease activity) used to treat complicated infections it interferes with peptidoglycan biosynthesis

Repurposing normal flor for better control

Modified Nissle 1917: A little bit of everything its a probiotic that is native to the GI tract. It helps prevent salmonella typhi and other such diseases

What are the sources of the following:

1. F6P

2. PEP

3. Gln

1. Glycolysis

2. Glycolysis

3. Primarily ingested