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Medical Microbiology > 3. The As, Bs, Cs and Ds of beta-lactamases > Flashcards

3. The As, Bs, Cs and Ds of beta-lactamases Flashcards

1. Describe ß-lactam antibiotics and how they work 2. Explain how PBPs can be modified to confer ß-lactam resistance 3. Define ß-lactamases and what they do 4. Describe the different types of ß-lactamases and how they differ 5. Give some examples of ß-lactamases

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1
Q

What is the structure of a gram-positive cell wall?

A
  1. A very thick layer of peptidoglycan that is anchored to the plasma membrane
  2. 1 lipid bilayer
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2
Q

What is the structure of a gram-negative cell wall?

A
  1. 2 lipid bilayers with a thin section of peptidoglycan between them.
  2. The space between the membranes is the periplasmic space
  3. The outer membrane makes gram-negative bacteria hard to treat as small molecules find it hard to penetrate the hydrophobic bilayer.
  4. The outer membrane contains porins for the passage of small molecules, mainly metabolites uptake, but this is how antibiotics get in
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3
Q

What makes up peptidoglycan?

A

2 sugars:
NAG - N-acetyl glucosamine
NAM - N-acetylmuramic acid
They are linked together by the peptide chains attached to NAM.

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4
Q

What are Penicillin Binding Proteins?

A

They are the enzymes that catalyse the formation of the peptide cross links in the formation of peptidoglycan.

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5
Q

What are the 2 reactions in the formation of peptidoglycan?

A
  1. transglycoslyase reaction
  2. Transpeptidase reaction
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6
Q

What happens in the transglycoslyase reaction?

A
  1. the NAG and NAM sugars are linked together.
  2. A free OH on the NAG attacks a carbon on the NAM.
  3. These sugars are continuously linked throughout the peptide.
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7
Q

What happens in the transpeptidase reaction?

A
  1. This links the peptide chains on the NAM sugars together by forming a covalent bond.
  2. The pentapeptide chain on the NAM contains a lysine and ends in 2 D-alanine residues.
  3. The amino group on the lysine attacks the C = O between the 2 D-alanines. This is a carbonyl bond.
  4. This breaks off a single terminal D-alanine and forms a cross link between 2 peptide chains.
  5. This reaction is repeated through the whole sheet of peptidoglycan.
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8
Q

What catalyses the transpeptidase reaction?

A

Penicillin Binding Proteins or PBPs

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9
Q

Why are PBPs called PBPs?

A
  1. They were 1st identified in the 80s based on their ability to bind penicillin.
  2. You could observe the PBPs using carbon-14 labelled penicillin.
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10
Q

What are the different types of PBPs??

A
  1. Low molecular weight PBPs.
  2. High molecular weight PBPs.
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11
Q

What are low molecular weight PBPs?

A
  1. about 40 KDa
  2. Only catalyse the transpeptidase reaction
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12
Q

What are high molecular weight PBPs?

A
  1. > 100KDa
  2. 2 classes
  3. Class A can catalyse both transglycosylase and transpeptidase reactions
  4. Class B only catalyses the transpeptidase reaction.
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13
Q

Why can class A high molecular weight PBPs catalyse both reactions?

A

Due to having 2 catalytic domains

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14
Q

Why are ß-lactams important?

A

They make up over 50% of prescribed antibiotics.

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15
Q

How do penicillins work?

A
  1. They are structural analogues of the D-ala D-ala terminal chain.
  2. This means they can enter the PBP active site and prevent its function..
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16
Q

What are the important features of penicillins?

A
  1. They contain a 4 membered ß-lactam ring.
  2. They also contain a carbonyl bond in the same place as D-ala.
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17
Q

What is the reaction mechanism of PBPs with D-ala-D-ala?

A
  1. The 2 D-ala residues bind into the active site of the transpeptidase domain of the PBP.
  2. In the active site, there is a nucleophilic serine that can donate a pair of electrons.
  3. The nucleophilic serine attacks the carbonyl bond between the 2 D-ala residues.
  4. This releases the terminal D-ala.
  5. An acyl intermediate is formed where the carbonyl group covalently links the peptide to the serine in the PBP active site.
  6. Attack from the amine group from the lysine on the carbonyl bond in the acyl-enzyme intermediate releases the covalent link with the serine and forms a cross-link between the amino acids.
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18
Q

What is the reaction mechanism of PBPs with penicillin?

A
  1. Acts in a similar way to the D-ala residues.
  2. The nucleophilic serine attacks the carbonyl group in the ß-lactam ring.
  3. A covalent link forms between the PBP active site serine and the ß-lactam ring in the penicillin.
  4. This breaks open the ß-lactam ring.
  5. This binding is very stable so the penicillin stays in the active site of the PBP causing a strong inhibition of the PBP transpeptidase domain.
  6. The D-ala then cannot bind into the PBP and peptidoglycan cannot be made and the cells die.
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19
Q

What are the 5 classes of ß-lactam antibiotics?

A
  1. Penicillins
  2. Cephalosporins
  3. Carbapenems
  4. Monobactams
  5. Penems
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20
Q

What do the different classes of ß-lactams have in common?

A

The ß-lactam ring with the carbonyl bond but they all have different attachments to the ring.

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21
Q

Why were Penems invented?

A
  1. They combine penicillins and cephalosporins.
  2. Developed in the 70s
  3. They are a totally synthetic class of antibiotics, whereas the rest are derived from natural products.
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22
Q

What are the major resistance mechanisms to ß-lactams?

A
  1. Target site modification of PBP to prevent ß-lactam binding.
  2. Inactivation of antibiotic through enzymes.
  3. Removal of antibiotic through efflux pumps.
  4. Altering the membrane permeability to impair antibiotic uptake.
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23
Q

How does MRSA acquire resistance to methicillins?

A
  1. It gained resistance 2/3 years to methicillin after its introduction.
  2. Methicillin inhibits PBPs 1, 2, 3 and 4.
  3. MecA is on a mobile genetic element that encodes PBP2 version called PBP2a.
  4. PBP2a has a low affinity to methicillin.
  5. This allows PBP2a to continue to carry out the transpeptidase reaction and make peptidoglycan.
  6. The MecA gene cassette inserts itself into the genome, causing a sudden gain of resistance.
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24
Q

What does methicillin inhibit?

A

PBPs 1, 2, 3 and 4

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25
Q

Where did MecA originate from?

A

S. fleurettii

26
Q

How does penicillin bind to PBPs?

A
  1. Penicillin binds to the PBP and forms a Michaelis complex.
  2. Then in 2nd step of the reaction the serine forms the covalent link with the penicillin in the active site of the transpeptidase.
  3. The 3rd step of the reaction is the hydrolysis of penicillin out from the PBP active site.
  4. However this step rarely ever happens due to the stability of the binding of penicillin and PBPs so the reaction stops with the penicillin in the PBP active site.
27
Q

How does PBP2a from MecA avoid methicillin?

A
  1. Methicillin doesn’t bind well to PBP2a due to a conformational change.
  2. This causes slow binding and acylation of the PBP2a active site compared to PBP2.
  3. Therefore very high concentrations of methicillin are needed to be effective and this is not clinically viable.
28
Q

What conformational change is present in PBP2a and how does this effect methicillin binding?

A
  1. The active site is covered so the binding of methicillin requires large structural changes.
  2. These changes are energetically unfavourable so they are unlikely to happen.
  3. This means methicillin is unlikely to bind.
  4. In order to force binding you need a large dose of methicillin
29
Q

How does PBP2a carry out the transpeptidase reaction if the active site is covered?

A
  1. Due to allosteric binding.
  2. A peptidoglycan analogue binds in the linker domain,
  3. A network of salt bridges connects this allosteric site to the active site.
  4. This opens the active site to allow binding of peptidoglycan D-ala-D-ala.
30
Q

How can we use allostery to target PBP2a and treat resistant MRSA infections?

A
  1. A cephalosporin antibiotic called ceftaroline can also bind an allosteric site on PBP2a.
  2. This site is in the linker but different to the peptidoglycan analogue binding site.
  3. This causes structural changes that causes the active site to open and another molecule of ceftaroline can bind.
31
Q

What are ß-lactamases?

A
  1. An enzyme that can degrade ß-lactam antibiotics.
  2. They bind to ß-lactams the same way PBPs do using a serine or metal ion.
  3. The 3rd reaction step of hydrolysis occurs very quickly, breaking the ß-lactam ring and making the antibiotic inactive.
32
Q

What do ß-lactamases do?

A

They break down the ß-lactam ring in the antibiotic so it cannot bind to PBPs and inhibit their function.

33
Q

What are the 2 groups of ß-lactamses?

A
  1. Serine ß-lactamases: Classes A, C, D
  2. Metallo ß-lactamases: Class B
34
Q

Examples of ß-lactamases: Class A

A

KPC-2
CTX-M
TEM-1

35
Q

Examples of ß-lactamases: Class C

A

AmpC

36
Q

Examples of ß-lactamases: Class D

A

OXA-48

37
Q

Examples of ß-lactamases: Class B

A

Splits into 3 subgroups
B1: NDM-1 - most relavent
B2: SphA
B3: L1

38
Q

How are serine ß-lactamases and PBPs related?

A
  1. Serine ß-lactamases evolved from PBP transpeptidase domains.
  2. Structurally very similar so they are almost identical.
  3. Serine in both active sites
  4. Do almost the same reactions
39
Q

What is the catalytic motif of the serine ß-lactamses and transpeptidase domains?

A

SXXK
Serine-non conserved- non conserved- lysine

40
Q

What is the acylation mechanism of action of ß-lactamases?

A
  1. The general base accepts a proton from the serine nucleophile. This activates the serins by putting a negative charge on the oxygen.
  2. This allows the O- to attack the carbonyl bond on the ß-lactam ring.
  3. The attack on the carbonyl bond forms a tetrahedral transition state which is very unstable.
  4. This intermediate breaks down by reaccepting a proton from the general base in the active site.
  5. This forms the acyl-enzyme intermediate where the ß-lactam ring is broken, and there is a covalent link between it and the serine in the active site of the ß-lactamase.
  6. Next deacylation occurs.
41
Q

What is a general base?

A

An amino acid that can accept a proton. In this context it accepts a proton from the serine nucleophile.

42
Q

What is the deacylation mechanism of action of ß-lactamases?

A
  1. The general base in the ß-lactamase active site accepts a proton from water.
  2. This generates an OH- ion from the water, and it can attack the carbonyl bond between the serine and the penicillin in the acyl-enzyme intermediate.
  3. This creates an unstable tetrahedral transition intermediate.
  4. This intermediate breaks down by reaccepting a proton from the general base.
  5. This causes the broken antibiotic to leave the active site of the ß-lactamase, and the active site regenerates as it was at the beginning.
43
Q

how do the different classes of serine ß-lactams differ?

A

They have different general bases in their active site

44
Q

What is the Class A ß-lactamase general base?

A

Glu - 166 (glutamate)

45
Q

What is the Class C ß-lactamase general base?

A

Lys-67 acting in conjugate with tyr-150

46
Q

What is the Class D ß-lactamase general base?

A

A carboxylated lysine - KCX70

47
Q

What is the class A ß-lactamase mechanism of action?

A
  1. Glu-166 accepts a proton from a water molecule to make an OH- group.
  2. The OH- accepts a proton from the nucleophilic serine to activate it.
  3. THe serine is then free to attack the carbonyl bond in the ß-lactam ring.
  4. This forms the unstable intermediate which is brokedn down by accepting a proton through ser130 and lys73 and finally accepting a proton back from Glu-166.
  5. Inactivated antibiotic is released.
48
Q

What is the class C ß-lactams mechanism of action?

A
  1. Lys67 accepts a proton from ser64 in the active site and generates a negative charge on the O.
  2. The unstable intermediate is formed.
  3. This breaks down by reaccepting a proton from lys67 and inactivating the antibiotic.
  4. Possible use of tyr150 that accepts a proton from water to generate an OH- group in the final step
49
Q

What is the class D ß-lactamase mechanism of action?

A
  1. A lysine with an added carboxylate group behaves like glu166 in class A enzymes.
  2. The carboyxlate group on KCX70 accetps a proton from the serine to generate O-.
  3. O- attacks the carbonyl bond.
  4. The unstable intermediate is formed and breaks down by reaccepting a proton from KCX70.
  5. The deacylation is similar to class A as the Carboxylate groups accept a proton from water to make OH- that attacks the carbonyl bond to release the inactive antibiotic.
50
Q

What is the common stage between all classes of ß-lacatamases?

A

The tetrahedral intermediate

51
Q

What are class B ß-lactamases?

A
  1. Metallo ß-lactamases
  2. Contain a Zn2+ or other available metal ion in the active site.
  3. The different subclasses have different amino acid make ups to change how the ion sits in the enzyme.
52
Q

B1 Metallo-ß-lactamases

A
  1. Contain 2 metal ions in the active site
  2. found on plasmids so spread quickly
53
Q

B2 Metallo-ß-lactamases

A
  1. Contains 1 metal ion in the active site
  2. Found on the chromosome
54
Q

B3 Metallo-ß-lactamases

A
  1. Contains 2 metal ions in the active site
  2. Found on the chromosome
55
Q

What is S. maltophilia?

A
  1. An important pathogen especially in the US.
  2. Produces a B3 Metallo-ß-lactamase, serine ß-lactamases and efflux pumps.
  3. Largly infects immunosuppressed patients
  4. Hard to treat
56
Q

What is the class B ß-lactams mechanism of action?

A
  1. The metal ions activates the water to become an OH-.
  2. The OH- attacks the carbonyl bond in the ß-lactam ring. No covalent link is formed.
  3. An unstable tetrahedral intermediate is formed and quickly breaks down to release the active antibiotic
57
Q

Important Class A ß-lactamases

A

The most widely distributed
BlaZ
TEM, SHV, CTX-M (all ESBLs)
KPC

58
Q

Important Class B ß-lactamases

A

Broad-spectrum and plasmid encoded
IMP
VIM
NDM1

59
Q

Important Class C ß-lactamases

A

AmpC
CMY

60
Q

Important Class D ß-lactamases

A

OXA enzymes

Medical Microbiology (17 decks)

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