A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to “skid” on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.
s –1), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. PBP3 performs substrate acylation in a highly efficient manner ( k cat/K m = 50,500 m –1 As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel β-sheet surrounded by α-helices. domains (Penicillin-binding protein And Serine/Threonine kinase. PBP3 folds into an NH 2-terminal, d, d-carboxypeptidase-like domain, and a COOH-terminal, elongated β-rich region. PBP1a (Ropp, et al., 2002) and PBP2 is homologous to E. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. that PBP3, previously shown to be dispensable, took on an essential role. The single d-Ala, d-Ala ( d, d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Remarkably, substitution of the catalytic serine of PBP2b had almost no effect. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. Furthermore, we explore temocillin's mechanism of β-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine β-lactamase. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. aeruginosa We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant ( k off ) in biochemical assays comparing temocillin with ticarcillin. Identification of accurate catalytic binding site was difficult because the target. Thus, recognizing the catalytic binding site residues in the protein structure was of high importance in computer-aided drug designing. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. The catalytic binding site was believed to be a small region, a cleft or pocket, where lead molecules can bind to stimulate the target protein and produce the desirable effect. The catalytic domain contains a serine (S307) that becomes transiently. aeruginosa have remained relatively unexplored. FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for. However, as described in detail above, we predict that the mutations P311S and V545I induce a more open catalytic site, and we hypothesize that the effect of this is to increase the. None of the mutations identified in PBP3 introduce a new catalytic residue in this region. Focusing only on positioning of active site residues to generate a nucleophilic serine. The origins of temocillin's inferior antibacterial properties against P. The proposed catalytic residues are located in loops at the active site or in the same helix as the reactive serine. De novo enzyme designs have generally tried to optimize multiple aspects of enzyme function simultaneously. 
The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against P. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older "forgotten" drugs, such as temocillin, for their ability to combat resistant microbes.
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