Penicillin-binding proteins (PBPs) are enzymes crucial for peptidoglycan biosynthesis in bacteria. Recent structural and biochemical studies have significantly advanced our understanding of PBPs' roles in peptidoglycan synthesis, maturation, and recycling. PBPs are classified into high molecular mass (HMM) and low molecular mass (LMM) categories, with HMM PBPs responsible for peptidoglycan polymerization and insertion into the cell wall, and LMM PBPs involved in cell separation, peptidoglycan maturation, and recycling. The PB domain, which shares structural similarities with penicillin, is essential for the D-peptidase activity of PBPs, catalyzing the hydrolysis of peptidoglycan cross-bridges. The transpeptidase mechanism involves the formation of an acyl-enzyme intermediate, followed by deacylation and cross-linking. β-lactam antibiotics inhibit PBPs by forming stable acyl-enzyme complexes. Class A PBPs, including monofunctional glycosyltransferases (MGTs), are responsible for peptidoglycan elongation, while class B PBPs, such as PBP2 and PBP3, are involved in cell division and elongation. The role of PBPs in peptidoglycan synthesis is essential for bacterial survival and shape maintenance.Penicillin-binding proteins (PBPs) are enzymes crucial for peptidoglycan biosynthesis in bacteria. Recent structural and biochemical studies have significantly advanced our understanding of PBPs' roles in peptidoglycan synthesis, maturation, and recycling. PBPs are classified into high molecular mass (HMM) and low molecular mass (LMM) categories, with HMM PBPs responsible for peptidoglycan polymerization and insertion into the cell wall, and LMM PBPs involved in cell separation, peptidoglycan maturation, and recycling. The PB domain, which shares structural similarities with penicillin, is essential for the D-peptidase activity of PBPs, catalyzing the hydrolysis of peptidoglycan cross-bridges. The transpeptidase mechanism involves the formation of an acyl-enzyme intermediate, followed by deacylation and cross-linking. β-lactam antibiotics inhibit PBPs by forming stable acyl-enzyme complexes. Class A PBPs, including monofunctional glycosyltransferases (MGTs), are responsible for peptidoglycan elongation, while class B PBPs, such as PBP2 and PBP3, are involved in cell division and elongation. The role of PBPs in peptidoglycan synthesis is essential for bacterial survival and shape maintenance.