Sandbox 128

Introduction

Peptidoglycan transpeptidase (TP) also known as penicillin-binding proteins (PBP), are essential for bacterial cell wall synthesis and catalyze the cross-linking of peptidoglycan polymers during bacterial wall synthesis.Beta-lactam antibiotic, which includes the penicillins,cephalosporins,carbapenems, and the monobactam aztreonam, bind and irreversibly inhibit the active site of TP. The overuse and misuse of b-lactam antibiotics has led to strains of Staphylococcus aureus (S.aureus) that are resistant to all currently available b-lactams and are often susceptible to so-called "last resort antibiotics", such as vancomycin.

Bacterial Cell Wall Structure

The bacterial cell wall is crucial for maintaining the structural integrity of bacteria and protects bacteria from osmotic stress and toxic compound. The cell wall is composed of peptidoglycan (Figure 2), and in Gram positive bacterial species (e.g. S. aureus) is many layers thick, while in Gram negative bacterial species (e.g. Escherichia coli) is only a few layers thick. The difference in the number of peptidoglycan layers accounts for the differential staining of these two groups of organisms. Peptidoglycan consists of a carbohydrate portion: alternating residues of N-acetylmuramic Acid (NAM) and N-acetylglucosamine (NAG) that polymerize to form long chains, and a protein portion: a pentapeptide chain that terminates with to D-alanines (D-Ala) residues. The pentapeptide chains are covalently bound to each NAM residue. Rows of peptidoglycan are cross-linked together with pentaglycine chains to form a "mesh-like" structure. This cross-linking reaction is catalyzed by TPs.

Figure 1.(A)Rows of Peptidoglycans forming a Bacterial Cell Wall (B)Peptidoglycan with D-Ala-D-Ala substrate

Catalytic Mechanism of Action of Transpeptidases

(a)The D-Ala side-chain substrate accesses the TP active site. (b)The active site serine residue nucleophilically attacks the peptide bond between the terminal D-Ala residues. Terminal D-Ala residue exits the active site, and the remaining D-Ala residue forms a covalent bond with the active site serine residue to form an acyl-TP complex. (c)Subsequently, a pentaglycine chain enters the TP active site through nucleophillic attack forms a covalent bond with the D-Ala formerly bound to the active site serine residue. As a result, the TP site serine residue is regenerated. The entire process takes approximately 4 seconds.

Figure 2. Schematic showing Catalytic Mechanism of PBP2a

Mechanism of Action of B-Lactam antibiotics

The B-Lactam antibiotics inhibit bacterial cell growth by irreversibly inhibiting TP's and, therefore, bacterial cell wall sythesis. Specifically, B-Lactams are molecular mimics of a portion of the peptidoglycan polymer, namely the D-Ala-D-Ala moiety, which is the normal TP enzymatic substrate(Figure 4). As a result, bacterial TP enzymes are "tricked" into reacting with B-Lactams. Additionally, the B-Lactams are very reactive molecules due to their B-lactam ring, and readily react with the TP active site serine residue and sterically block the active site preventing the entry of nucleophiles that regenerate the active site serine residue such as the pentaglycine chain or water.

Structure of PBP2a, a B-Lactam Resistant Transpeptidase