Penicillin-binding proteins (PBPs) are essential enzymes in bacterial peptidoglycan biosynthesis, involved in the polymerization and cross-linking of glycan chains. PBPs are classified into high molecular mass (HMM) and low molecular mass (LMM) categories, with HMM PBPs responsible for peptidoglycan synthesis and insertion into the cell wall, while LMM PBPs are involved in cell separation, maturation, and recycling. PBPs have a common DD-peptidase activity, including transpeptidase, carboxypeptidase, and endopeptidase functions. The PB domain of PBPs is responsible for binding β-lactam antibiotics, which inhibit their activity by mimicking the natural substrate of PBPs. Structural studies have revealed that the active site of PBPs contains conserved residues that are crucial for substrate binding and catalytic activity. The transpeptidase mechanism involves the acylation of the penultimate D-alanine of the donor strand, followed by deacylation, which can occur through hydrolysis or cross-link formation. The catalytic mechanism of PBPs involves a double lysine-serine system, with the active site containing conserved residues that are essential for the correct positioning of the substrate. PBPs are sensitive to β-lactam antibiotics, forming a stable acyl-enzyme complex that impairs their peptidoglycan cross-linking capability. The structural basis of PBPs includes a complex N-terminal domain and a C-terminal transpeptidase domain, with the N-terminal domain interacting with other proteins involved in cell division. Class B PBPs are involved in cell division and are responsible for the high level of resistance to β-lactam antibiotics in some bacteria. The role of class B PBPs in peptidoglycan biosynthesis is crucial for cell elongation, shape maintenance, and division. The resistance to β-lactams in many bacteria is due to the presence of penicillin-resistant PBPs that can take over the transpeptidase function of other PBPs. The structural and functional diversity of PBPs across different bacteria highlights their importance in bacterial survival and adaptation to environmental stresses.Penicillin-binding proteins (PBPs) are essential enzymes in bacterial peptidoglycan biosynthesis, involved in the polymerization and cross-linking of glycan chains. PBPs are classified into high molecular mass (HMM) and low molecular mass (LMM) categories, with HMM PBPs responsible for peptidoglycan synthesis and insertion into the cell wall, while LMM PBPs are involved in cell separation, maturation, and recycling. PBPs have a common DD-peptidase activity, including transpeptidase, carboxypeptidase, and endopeptidase functions. The PB domain of PBPs is responsible for binding β-lactam antibiotics, which inhibit their activity by mimicking the natural substrate of PBPs. Structural studies have revealed that the active site of PBPs contains conserved residues that are crucial for substrate binding and catalytic activity. The transpeptidase mechanism involves the acylation of the penultimate D-alanine of the donor strand, followed by deacylation, which can occur through hydrolysis or cross-link formation. The catalytic mechanism of PBPs involves a double lysine-serine system, with the active site containing conserved residues that are essential for the correct positioning of the substrate. PBPs are sensitive to β-lactam antibiotics, forming a stable acyl-enzyme complex that impairs their peptidoglycan cross-linking capability. The structural basis of PBPs includes a complex N-terminal domain and a C-terminal transpeptidase domain, with the N-terminal domain interacting with other proteins involved in cell division. Class B PBPs are involved in cell division and are responsible for the high level of resistance to β-lactam antibiotics in some bacteria. The role of class B PBPs in peptidoglycan biosynthesis is crucial for cell elongation, shape maintenance, and division. The resistance to β-lactams in many bacteria is due to the presence of penicillin-resistant PBPs that can take over the transpeptidase function of other PBPs. The structural and functional diversity of PBPs across different bacteria highlights their importance in bacterial survival and adaptation to environmental stresses.