Sandbox 126

<StructureSection load='4dki' size=500 side=right scene='37/372726/Pbp2a_with_residues/3'>

Transpeptidases (TP), also known as penicillin-binding proteins (PBP), catalyze the cross-linking of peptidoglycan polymers during bacterial cell wall synthesis. Beta-lactam (β-lactam) antibiotics, which include penicillins, cephalosporins and carbapenems, bind and irreversibly inhibit transpeptidases. The overuse and misuse of β-lactam antibiotics has led to strains of Staphylococcus aureus that are resistant to all β-lactams and are often only susceptible to “last resort antibiotics”, such as vancomycin.

== 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 are 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.

[[Image:Cell Wall 7 30 2013.jpg|thumb|alt= Alt text| Figure 1. A.Bacterial Cell Wall B.Peptidoglycan with D-Ala-D-Ala substrate B. Peptidoglycan Unit |550px]]

== Structure of a Resistant Transpeptidase ==

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='37/372726/Tp_and_npb_domain/3'>a non-penicillin binding (NPB) domain and a TP domain</scene>. 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 <scene name='37/372726/Close_up_of_active_site/8'>active site</scene> contains a serine residue at position <scene name='37/372726/Ser403_no_backbone/1'>403</scene> (Ser403), which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.

== Catalytic Mechanism of PBP2a ==

[[Image:Schematic TP 3steps.jpg|thumb|alt= Alt text|Figure 2. Schematic diagram illustrating the mechanism of action of PBP2a |550px]]

(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.

The entire process takes 4 milliseconds.

== 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.

[[Image:Structures on penicillin and b lactam.jpg|thumb|alt= Alt text|Figure 3. Mechanism of action of β-lactams. A. Structure of a β-lactam (penicillin) showing the amide, carboxyl, and β-lactam ring groups β-lactam ring groups. B. Structure of the D-Ala-D-Ala substrate. C. Overlay of the D-Ala-D-Ala substrate in red with penicillin demonstrating molecular mimicry.|550 px]]

== 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 – ceftobiprole and

ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to

inhibit PBP2a because additional chemical groups at the <scene name='37/372726/Ceftobiprole/4'>R2</scene> position of the cephalosporin

backbone are able to interact with additional amino acid residues in PBP2a; specifically

<scene name='37/372726/Met641_and_tyr446/1'>Tyr446 and Met641</scene>. As a result of its <scene name='37/372726/Active_site_with_ceftobiprole/2'>tighter binding to PBP2a</scene>, ceftobiprole is able to more efficiently react with the serine active site residue and therefore inhibit the activity of PBP2a.

Figure 5. Mechanism of action of ceftobiprole. (a) Structure of ceftobriprole.

(b) The R2 group of ceftobiprole is bound by PBP2a, Tyr446, and Met641. This

increased binding allows the serine residue active site (Ser403) to hydrolyze the β-

lactam ring and become irreversibly inhibited. (c) Schematic of β-lactam covalently

bonded to active site blocking entrance to the substrate.

== PBP2a and Ceftaroline ==

<scene name='37/372726/3zfz_with_muramic_acid/1'>PBP2a in complex with Ceftaroline (3zfz)</scene>

In addition to TP domain of PBP2a, there is an allosteric domain in which the distance between the the active site and the allosteric site is 60Å. Allosteric site serves as a binding site for the substrate peptidoglycan. When the substrate binds to the <scene name='37/372726/3zfz_allosteric_site/1'>allosteric site</scene>, a conformational change occurs at the active site, opening it and allowing catalytic action to occur. The medicine,<scene name='37/372726/Ceftaroline/3'>Ceftaroline</scene>, <scene name='37/372726/3zfz_allosteric_site_and_ceft/1'>mimics the substrate at the allosteric site</scene> opening the active site, allowing ceftaroline to enter and bind noncovalently.