Structural analysis of an “open” form of PBP1B from <i>Streptococcus pneumoniae</i>

Lovering, Andrew L.; Castro, Liza de; Lim, Daniel; Strynadka, N.C.J.

doi:10.1110/ps.062112106

Cited by 28 publications

(33 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The induced-fit conformational change from the uncomplexed PBP 2X is also expected to be required for substrate binding, in which a hydrogen bond forms between the amido moiety of the substrate, Thr550 and Asn397. Previous structural analyses of S. pneumoniae PBP 1B have revealed not only the "closed" active-site conformation but also the "open" active-site conformation in the absence of ␤-lactam antibiotics (16). In conclusion, the C-7 side chains of cephalosporins apparently contribute high-affinity interactions with PBPs by mimicking the donor strand, and they also take advantage of flexibility in the active site to accommodate the substrate.…”

Section: Resultsmentioning

confidence: 81%

Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

Yamada

Watanabe

Miyara

et al. 2007

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Cefditoren is the active form of cefditoren pivoxil, an oral cephalosporin antibiotic used for the treatment of respiratory tract infections and otitis media caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, and methicillin-susceptible strains of Staphylococcus aureus. ␤-Lactam antibiotics, including cefditoren, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. To envision the binding of cefditoren to PBPs, we determined the crystal structure of a trypsin-digested form of PBP 2X from S. pneumoniae strain R6 complexed with cefditoren. There are two PBP 2X molecules (designated molecules 1 and 2) per asymmetric unit. The structure reveals that the orientation of Trp374 in each molecule changes in a different way upon the formation of the complex, but each forms a hydrophobic pocket. The methylthiazole group of the C-3 side chain of cefditoren fits into this binding pocket, which consists of residues His394, Trp374, and Thr526 in molecule 1 and residues His394, Asp375, and Thr526 in molecule 2. The formation of the complex is also accompanied by an induced-fit conformational change of the enzyme in the pocket to which the C-7 side chain of cefditoren binds. These features likely play a role in the high level of activity of cefditoren against S. pneumoniae.

show abstract

Section: Resultsmentioning

confidence: 81%

Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

Yamada

Watanabe

Miyara

et al. 2007

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…An N-terminal thioredoxin (Trx)-His 6 fusion protein lacking the transmembrane helix could be expressed in the cytoplasm but underwent proteolysis in the transpeptidase domain. Because crystal structures already exist for the C-terminal transpeptidase domain of orthologs of A. aeolicus PBP1A (7,12,13), we focused on identifying a well behaved PGT domain. Several PGT constructs of varying lengths were prepared and analyzed to establish the minimum size of the PGT domain.…”

Section: Resultsmentioning

confidence: 99%

“…Major advances have been made in recent years with respect to the characterization of key biosynthetic enzymes (3)(4)(5). Several structures have been reported for enzymes that catalyze transpeptidation (6,7), but no PGT structures have been described.…”

mentioning

confidence: 99%

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Yuan

Barrett²,

Zhang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

138

122

View full text Add to dashboard Cite

Peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. Peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the ␤-lactams, imipenems, cephalosporins, and glycopeptides. Resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. Representative structures have been reported for most of the enzymes in the pathway. Until now, however, there have been no structures of any peptidoglycan glycosyltransferases (also known as transglycosylases), which catalyze formation of the carbohydrate chains of peptidoglycan from disaccharide subunits on the bacterial cell surface. We report here the 2.1-Å crystal structure of the peptidoglycan glycosyltransferase (PGT) domain of Aquifex aeolicus PBP1A. The structure has a different fold from all other glycosyltransferase structures reported to date, but it bears some resemblance to -lysozyme, an enzyme that degrades the carbohydrate chains of peptidoglycan. An analysis of the structure, combined with biochemical information showing that these enzymes are processive, suggests a model for glycan chain polymerization.antibiotic resistance ͉ penicillin-binding protein ͉ cell wall ͉ transglycosylase T he major component of the bacterial cell wall is a cross-linked glycopeptide polymer called peptidoglycan. This polymer surrounds the cytoplasmic membrane of bacteria and functions as an exoskeleton, maintaining cell shape and stabilizing the membrane against fluctuations in osmotic pressure. Peptidoglycan is synthesized in an intracellular phase in which UDP-Nacetylglucosamine is converted to a diphospholipid-linked disaccharide-pentapeptide known as Lipid II, and in an extracellular phase in which the disaccharide (NAG-NAM) subunits of translocated Lipid II are coupled by peptidoglycan glycosyltransferases (PGTs; also known as transglycosylases) to form linear carbohydrate chains ( Fig. 1), which are cross-linked through the attached peptide moieties by transpeptidases (1). A functioning peptidoglycan pathway is required for bacterial cell growth and division, and compounds that inhibit peptidoglycan biosynthesis have antibiotic activity (2). For example, the ␤-lactams, which have been used for decades to treat bacterial infections, irreversibly inhibit the transpeptidases. The emergence of resistance to ␤-lactams and other clinically used antibiotics has prompted intense interest in understanding peptidoglycan biosynthesis in detail. Major advances have been made in recent years with respect to the characterization of key biosynthetic enzymes (3-5). Several structures have been reported for enzymes that catalyze transpeptidation (6, 7), but no PGT structures have been described.PGTs are defined by the presence of five conserved sequence motifs ( Fig. 2A) and exist in two forms: (i) as N-terminal glycosyltransferase domains in bifunctional proteins that also contain a C-terminal transpeptidase domain [called class A penicillin-bindi...

show abstract

“…SpPBP 1b harbors a tryptophan (W517) in the middle position of the SXN motif, and this aromatic amino acid forms two interactions with residues in the loop: 1) a π-π interaction with the phenolic side chain of Tyr498, and 2) a carboxamide-π interaction with Asn485 (Fig. 5B) (46, 47). Lastly, SaPBP 2 has an SFN motif where Phe455 forms a π-π interaction with the loop residue His442, which is held in place by interactions of the imidazolium nitrogens with two neighboring aspartate residues (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Highly Conserved Interaction Involving the Middle Residue of the SXN Active-Site Motif Is Crucial for Function of Class B Penicillin-Binding Proteins: Mutational and Computational Analysis of PBP 2 from N. gonorrhoeae

Tomberg¹,

Temple

Fedarovich

et al. 2012

Biochemistry

View full text Add to dashboard Cite

Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs and its importance is also supported by multi-sequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g. by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

show abstract

Structural analysis of an “open” form of PBP1B from Streptococcus pneumoniae

Cited by 28 publications

References 22 publications

Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

A Highly Conserved Interaction Involving the Middle Residue of the SXN Active-Site Motif Is Crucial for Function of Class B Penicillin-Binding Proteins: Mutational and Computational Analysis of PBP 2 from N. gonorrhoeae

Contact Info

Product

Resources

About