Crystal Structures of Penicillin-Binding Protein 3 from Pseudomonas aeruginosa: Comparison of Native and Antibiotic-Bound Forms

Sainsbury, Sarah; Bird, Louise E.; Rao, V.A.; Shepherd, Sharon M.; Stuart, David I.; Hunter, William N.; Owens, Raymond J.; Ren, Jingshan

doi:10.1016/j.jmb.2010.10.024

Cited by 80 publications

(92 citation statements)

References 37 publications

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“…The oxygen atom of the tartrate carboxylate group occupies the oxyanion hole by forming hydrogen bonds with the main-chain nitrogen atoms of Ser394 and Ser578. In general, the oxyanion hole is occupied by the ␤-lactam carbonyl oxygen in the acylated PBPs (28,29). Inter- estingly, polycarboxylates, such as citrate or tartrate, have been observed in the active sites of other ␤-lactamases, such as the class A carbapenemase KPC-2 (30), the plasmid-encoded class C ␤-lactamase CMY-2 (PDB code 1ZC2), a metallo-␤-lactamase (PDB code 1MQO) for citrate, and the class D ␤-lactamase OXA-46 (31) for tartrate.…”

Section: Resultsmentioning

confidence: 99%

“…4). In general, the carboxylate group hydrogen bonds with a serine or threonine residue immediately following the KTG motif of the ␤3 strand, and the interaction probably contributes to recovering the antiparallel nature of ␤3 and ␤4 in the acylated form (29,32). Interestingly, the conformation of ␤3 in the apo-form of LmPBP4 is stably positioned in antiparallel fashion to ␤4, and the loop connecting the two strands is located in the open conformation of the active-site groove.…”

Section: Resultsmentioning

confidence: 99%

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Crystal Structures of Bifunctional Penicillin-Binding Protein 4 from Listeria monocytogenes

Jeong

Kim

Rojviriya

et al. 2013

Antimicrob Agents Chemother

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b Penicillin-binding proteins (PBPs), which catalyze the biosynthesis of the peptidoglycan chain of the bacterial cell wall, are the major molecular target of bacterial antibiotics. Here, we present the crystal structures of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase (TP) PBP4 from Listeria monocytogenes in the apo-form and covalently linked to two ␤-lactam antibiotics, ampicillin and carbenicillin. The orientation of the TP domain with respect to the GT domain is distinct from that observed in the previously reported structures of bifunctional PBPs, suggesting interdomain flexibility. In this structure, the active site of the GT domain is occluded by the close apposition of the linker domain, which supports the hypothesis that interdomain flexibility is related to the regulation of GT activity. The acylated structures reveal the mode of action of ␤-lactam antibiotics toward the class A PBP4 from the human pathogen L. monocytogenes. Ampicillin and carbenicillin can access the active site and be acylated without requiring a structural rearrangement. In addition, the active site of the TP domain in the apoform is occupied by the tartrate molecule via extensive hydrogen bond interactions with the catalytically important residues; thus, derivatives of the tartrate molecule may be useful in the search for new antibiotics to inhibit PBPs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Crystal Structures of Bifunctional Penicillin-Binding Protein 4 from Listeria monocytogenes

Jeong

Kim

Rojviriya

et al. 2013

Antimicrob Agents Chemother

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“…Both had the same PBP3 mutation resulting in the change of R504C. This residue is part of an important hinge region of the PBP (22) and may cause interference with ceftazidime binding. These isolates were of different STs from India and the United States, strongly indicating independent acquisition of this amino acid variation.…”

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confidence: 99%

Elucidation of Mechanisms of Ceftazidime Resistance among Clinical Isolates of Pseudomonas aeruginosa by Using Genomic Data

Kos

McLaughlin

Gardner

2016

Antimicrob Agents Chemother

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Ceftazidime is one of the few cephalosporins with activity against Pseudomonas aeruginosa. Using whole-genome comparative analysis, we set out to determine the prevalent mechanism(s) of resistance to ceftazidime (CAZ) using a set of 181 clinical isolates. These isolates represented various multilocus sequence types that consisted of both ceftazidime-susceptible and -resistant populations. A presumptive resistance mechanism against ceftazidime was identified in 88% of the nonsusceptible isolates using this approach. Pseudomonas aeruginosa is an opportunistic pathogen associated with numerous nosocomial infections, where ␤-lactam antibiotics remain key in treatment (1, 2). One of the major antimicrobials used to fight P. aeruginosa infections is ceftazidime (CAZ), a well-known cephalosporin that acts primarily as a penicillin-binding protein 3 (PBP3) inhibitor (3,4).A significant proportion of ceftazidime-resistant isolates arise through the horizontal acquisition of ␤-lactamases or altered expression of the chromosomal drug-inducible wide-spectrum class C ␤-lactamase AmpC (reviewed in reference 5). The overproduction of AmpC can result from mutations affecting the peptidoglycan (PG) recycling process, where accumulation of cell wall intermediates ultimately induces ampC overexpression (5).We focused our study on a panel of 181 clinical P. aeruginosa isolates, where comparative analysis between multiple isolates belonging to the same multilocus sequence types (MLSTs) allowed for identification of chromosomal gene variants unique to the ceftazidime-resistant population (6).The initial MIC to ceftazidime was determined using frozen plates (Thermo Scientific) following the Clinical and Laboratory Standards Institute guidelines (7,8). Of the 181 isolates in the analysis set, 99 (55%) were resistant to ceftazidime (MIC, Ն16 g/ml), and 82 were susceptible (MIC, Յ8 g/ml) ( Table 1; see Table S1 in the supplemental material).Genomic analysis (see Table S1 in the supplemental material) was performed using CLC Genomic Workbench 7.0.4 (CLCBio). Unique sequence variants exclusive to the resistant population of each MLST group were flagged for further analysis as outlined in the summary column of Table S1. To account for resistance in the 99 ceftazidime-resistant isolates in a parsimonious manner, we followed a triage process-accounting first for resistance-inducing ␤-lactamases, second for mechanisms allowing for derepression of ampC, and third for other candidate causes of resistance.Forty-six isolates had ␤-lactamases that have been reported to hydrolyze ceftazidime (Table 1). Analysis of the ampC regulon and additional cephalosporin targets was also completed for these isolates (see Table S1 in the supplemental material); however, resistance was attributed primarily to the presence of the ␤-lactamases, as clinically, detection of such an element would rule out treatment with ceftazidime. It was apparent that certain MLST lineages were enriched in ␤-lactamases, particularly sequence type 111 (ST111) and ST233, which were t...

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“…The small adjacent N‐terminal segment of the transpeptidase domain comprises two α‐helices [α1n (250–268) and α2n (270–276)] and a three‐stranded [β1n (280–284), β2n (480–486), and β3n (490–495)] antiparallel β‐sheet (Figs 1A and 2). The PonA1 transpeptidase (or penicillin‐binding domain) resembles other PBPs secondary structure: PBP1 of class A from Staphylococcus pneumonia (RMSD of 1.9 Å) 24, 25, the transpeptidase domain of PBP2 from Neisseria gonorroeae (RMSD of 2.00 Å) 26, class A PBP1 from Pseudomonas aeruginosa (RMSD of 1.65 Å) 27, 28, and PBP4 from Listeria monocytogenes (RMSD of 2.01 Å) 29. A cleft in the middle of the domain contains the active site.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

et al. 2016

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Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections.DatabaseStructural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

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Crystal Structures of Penicillin-Binding Protein 3 from Pseudomonas aeruginosa: Comparison of Native and Antibiotic-Bound Forms

Cited by 80 publications

References 37 publications

Crystal Structures of Bifunctional Penicillin-Binding Protein 4 from Listeria monocytogenes

Crystal Structures of Bifunctional Penicillin-Binding Protein 4 from Listeria monocytogenes

Elucidation of Mechanisms of Ceftazidime Resistance among Clinical Isolates of Pseudomonas aeruginosa by Using Genomic Data

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

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