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==='''Introduction'''===
==='''Introduction'''===


<scene name='37/372724/Tp_1_in_grey/1'>Transpeptidase</scene>(TP),
<scene name='37/372724/Tp_1_in_grey/1'>Transpeptidase</scene>
also known as penicillin-binding proteins (PBP), catalyze the cross-linking of  
(TP), also known as penicillin-binding proteins  
peptidoglycan polymers during bacterial cell wall synthesis. The natural transpeptidase
(PBP), catalyze the cross-linking of peptidoglycan polymers during
substrate is the D-Ala-D-Ala peptidoglycan side chain terminus. Beta-lactam (β-lactam)
bacterial cell wall synthesis. Beta-lactam (β-lactam) antibiotics,
antibiotics, which include penicillins, cephalosporins and carbapenems, bind and  
which include penicillins, cephalosporins and carbapenems, bind and
irreversibly inhibit transpeptidases by mimicking the D-Ala-D-Ala substrate, resulting
irreversibly inhibit transpeptidases. The overuse and misuse of β-lactam
in the inhibition of cell wall synthesis and ultimately bacterial cell growth. Overuse
antibiotics has led to strains of Staphylococcus aureus that are resistant
and misuse of β-lactams has led to the generation of methicillin-resistant Staphylococcus  
to all β-lactams and are often only susceptible to “last resort antibiotics”,
aureus (MRSA) isolates that have acquired an alternative transpeptidase, PBP2a, which is
such as vancomycin.  
neither bound nor inhibited by β-lactams. MRSA isolates are resistant to all β-lactams, and
are often only susceptible to “last resort antibiotics”, such as vancomycin. Recently, two
cephalosporins - ceftobiprole and ceftaroline - that bind and inhibit PBP2a have been
developed.  




'''Cell Wall Synthesis'''
β-Lactam antibiotics stop the production of the cell wall by targeting bacterial
PBPs. The cell wall, which is composed of peptidoglycan and surrounds the cell
membrane, is crucial for maintaining the structural integrity of the bacterium.
The cell wall is composed of rows of peptidoglycan cross-linked together with
pentaglycine chains. Peptidoglycan consists of N-acetylmuramic Acid (NAM) and
N-acetylglucosamine (NAG) polymers. The NAM residues have a five amino acid side
chain that terminates with two D-Alanine (D-Ala) residues. MRSA is resistant to all
β-lactams because it acquires an alternative PBP, PBP2a, that is not bound or
inhibited by any β-lactams. Recently, two cephalosporins - ceftobiprole and
ceftaroline - that bind and inhibit PBP2a have been developed.'''


'''Cell Wall Structure'''
The cell wall, which is composed of peptidoglycan, is crucial for maintaining
the structural integrity of the bacterium.  Peptidoglycan consists of
N-acetylmuramic Acid (NAM) and N-acetylglucosamine (NAG) polymers. Rows of
peptidoglycan cross-linked together with pentaglycine chains. The NAM residues
have a five amino acid side chain that terminates with two D-Alanine (D-Ala)
residues.


==='''How does PBP2a works?'''===
PBP2a is composed of two domains: a <scene name='37/372724/Non_penicillin_binding/1'>non penicillin binding</scene> (NPB) domain and a
<scene name='37/372724/Transpeptidase_domain/1'>TP</scene> domain. The NBP domain of PBP2a is anchored in the cell membrane, while the TP domain “sits” in the periplasm with its active site facing the inner surface of the cell wall. The active site contains a
serine residue at position 403 (<scene name='37/372724/Serine403label/2'>ser403</scene>) which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.


'''Catalytic Mechanism of PBP2a
==='''Structure of a Resistant Transpeptidase'''===
The D-Ala-D-Ala side-chain substrate of the peptidoglycan accesses the active site of the PBP2a.
Methicillin resistant Staphylococcus aureus (MRSA) is resistant to all β-lactams because it acquires an alternative PBP, PBP2a, that is not bound or inhibited by any β-lactams. PBP2a is composed of two domains: <scene name='37/372724/non_penicillin_binding/1'>non penicillin binding</scene> (NPB) domain and a <scene name='37/372724/Transpeptidase_domain/1'>TP</scene> domain. The NBP domain of PBP2a is anchored in the cell membrane, while the TP domain “sits” in the periplasm with its active site facing the inner surface of the cell wall. The active site contains a serine residue at position 403 (<scene name='37/372724/Serine403label/2'>Ser403</scene>)which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.
Ser403 nucleophilically attacks the peptide bond of the terminal D-Ala residues of the substrate. The terminal D-Ala residue then exits the active site.
The now terminal D-Ala residue forms a covalent bond to Ser403, while a cross-linking pentaglycine chain enters the active site.
A covalent bond forms between the pentaglycine chain and the terminal D-Ala residue, regenerating the active site serine residue.




==='''Catalytic Mechanism of PBP2a'''===
(a) The D-Ala-D-Ala side-chain substrate of the peptidoglycan accesses the active site of
the PBP2a.
(b) Ser403 nucleophilically attacks the peptide bond of the terminal D-Ala residues of the
substrate. The terminal D-Ala residue then exits the active site.
(c) The now terminal D-Ala residue forms a covalent bond to Ser403, while a crosslinking
pentaglycine chain enters the active site.
(d) A covalent bond forms between the pentaglycine chain and the terminal D-Ala
residue, regenerating the active site serine residue.


==='''How does Ceftobiprole work?'''===
The entire process takes 4 milliseconds.
MRSA becomes resistant to β-lactams by acquiring an alternative PBP, PBP2a, that is neither bound nor inhibited by β-lactams. Recently, two cephalosporins – <scene name='37/372724/Ceftobiprole/1'>ceftobiprole</scene> and ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to inhibit PBP2a because additional chemical groups at the <scene name='37/372724/Ceftobiprole/2'>R2</scene> position of the cephalosporin backbone are able to interact with additional amino acid residues in PBP2a; specifically <scene name='37/372724/Met641_and_tyr446_labeled/1'>Tyr446 and Met641</scene>. As a result of its tighter binding to PBP2a,  
 
<scene name='37/372724/Rb6_interactions/1'>ceftobiprole</scene> is able to more efficiently react with the serine active site residue and therefore inhibit the activity of PBP2a.
==='''How do antibiotics work?'''===
The β-lactam antibiotics inhibit bacterial growth by inhibiting PBPs and ultimately cell wall
synthesis. Specifically, β-lactams are molecular mimics of D-Ala-D-Ala, which is the normal
substrate of PBPs. Nucleophillic attack of the β-lactam results in the PBP being irreversibly
inhibited by the β-lactam. As a result, the synthesis of the cell wall is inhibited which leads
to cell lysis.
 
==='''PBP2a and Ceftobiprole'''===
MRSA becomes resistant to β-lactams by acquiring an alternative PBP, PBP2a, that is
neither bound nor inhibited by β-lactams. Recently, two cephalosporins –  
<scene name='37/372724/Ceftobiprole/1'>ceftobiprole</scene> and
ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to
inhibit PBP2a because additional chemical groups at the <scene name='37/372724/Ceftobiprole/2'>R2</scene>
position of the cephalosporin backbone are able to interact with additional amino acid
residues in PBP2a; specifically <scene name='37/372724/Met641_and_tyr446_labeled/1'>Tyr446 and Met641</scene>.
As a result of its tighter binding to PBP2a, <scene name='37/372724/Rb6_interactions/1'>ceftobiprole</scene>
is able to more efficiently react with the serine active site residue and therefore inhibit the activity of PBP2a.

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