A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

Lee, Wenlin; McDonough, Michael A.; Kotra, Lakshmi P.; Li, Zhihong; Silvaggi, N.R.; Takeda, Yasuharu; Kelly, Judith A.; Mobashery, Shahriar

doi:10.1073/pnas.98.4.1427

Cited by 96 publications

(94 citation statements)

References 26 publications

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“…The amide groups of the -lactams, 10-13, in the AmpC/acyl-enzyme complexes made interactions characteristic of this site in -lactam-recognizing enzymes. 20,[30][31][32][33][34] Atom Nδ2 of Asn152 hydrogen bonds with the ligand amide oxygen in all of the complexes, although this distance is slightly long in the AmpC/13 complex (3.3 Å). Atom N 2 of Gln120 interacts with the ligand amide oxygen only in the AmpC/13 complex.…”

Section: Resultsmentioning

confidence: 99%

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

2002

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-Lactamases are the most widespread resistance mechanism to -lactam antibiotics and are an increasing menace to public health. Several -lactamase structures have been determined, making this enzyme an attractive target for structure-based drug design. To facilitate inhibitor design for the class C -lactamase AmpC, binding site "hot spots" on the enzyme were identified using experimental and computational approaches. Experimentally, X-ray crystal structures of AmpC in complexes with four boronic acid inhibitors and a higher resolution (1.72 Å) native apo structure were determined. Along with previously determined structures of AmpC in complexes with five other boronic acid inhibitors and four -lactams, consensus binding sites were identified. Computationally, the programs GRID, MCSS, and X-SITE were used to predict potential binding site hot spots on AmpC. Several consensus binding sites were identified from the crystal structures. An amide recognition site was identified by the interaction between the carbonyl oxygen in the R1 side chain of -lactams and the atom Nδ2 of the conserved Asn152. Surprisingly, this site also recognizes the aryl rings of arylboronic acids, appearing to form quadrupole-dipole interactions with Asn152. The highly conserved "oxyanion" hole defines a site that recognizes both carbonyl and hydroxyl groups. A hydroxyl binding site was identified by the O2 hydroxyl in the boronic acids, which hydrogen bonds with Tyr150 and a conserved water. A hydrophobic site is formed by Leu119 and Leu293. A carboxylate binding site was identified by the ubiquitous C3(4) carboxylate of the -lactams, which interacts with Asn346 and Arg349. Four water sites were identified by ordered waters observed in most of the structures; these waters form extensive hydrogen-bonding networks with AmpC and occasionally the ligand. Predictions by the computational programs showed some correlation with the experimentally observed binding sites. Several sites were not predicted, but novel binding sites were suggested. Taken together, a map of binding site hot spots found on AmpC, along with information on the functionality recognized at each site, was constructed. This map may be useful for structure-based inhibitor design against AmpC.

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Section: Resultsmentioning

confidence: 99%

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

2002

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“…We underscore that these conformational changes are expected to be operative during the typical turnover events by this enzyme as well in light of the closed nature of the active site. A volume in excess of 1000 Å 3 is needed for the sequestration of the two peptidoglycan residues within the active site for the transpeptidase activity (33). The requisite conformational change would be expected to create this space for the catalytic events.…”

Section: Resultsmentioning

confidence: 99%

The Basis for Resistance to β-Lactam Antibiotics by Penicillin-binding Protein 2a of Methicillin-resistant Staphylococcus aureus

Fuda

Suvorov

Vakulenko

et al. 2004

Journal of Biological Chemistry

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223

200

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Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available ␤-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several ␤-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by ␤-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k 2 ) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillinsensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (K d ) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.

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“…By inactivating peptidoglycan transpeptidases, members of the family of penicillin-binding proteins, ␤-lactam antibiotics have been successfully used for many years to inhibit the peptidoglycan synthesis responsible for the biosynthesis of the bacterial cell wall (1,2).…”

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confidence: 99%

Crystal structure of the carbapenemase OXA-24 reveals insights into the mechanism of carbapenem hydrolysis

Santillana

Beceiro

Bou

et al. 2007

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

109

162

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Combating bacterial resistance to ␤-lactams, the most widely used antibiotics, is an emergent and clinically important challenge. OXA-24 is a class D ␤-lactamase isolated from a multiresistant epidemic clinical strain of Acinetobacter baumannii. We have investigated how OXA-24 specifically hydrolyzes the last resort carbapenem antibiotic, and we have determined the crystal structure of OXA-24 at a resolution of 2.5 Å. The structure shows that the carbapenem's substrate specificity is determined by a hydrophobic barrier that is established through the specific arrangement of the Tyr-112 and Met-223 side chains, which define a tunnel-like entrance to the active site. The importance of these residues was further confirmed by mutagenesis studies. Biochemical and microbiological analyses of specific point mutants selected on the basis of structural criteria significantly reduced the catalytic efficiency (kcat/Km) against carbapenems, whereas the specificity for oxacillin was noticeably increased. This is the previously unrecognized crystal structure that has been obtained for a class D carbapenemase enzyme. Accordingly, this information may help to improve the development of effective new drugs to combat ␤-lactam resistance. More specifically, it may help to overcome carbapenem resistance in A. baumannii, probably one of the most worrying infectious threats in hospitals worldwide.␤-lactamases ͉ carbapenem resistance ͉ enzyme mechanism ͉ protein crystallography ͉ Acinetobacter baumannii

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A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

Cited by 96 publications

References 26 publications

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

The Basis for Resistance to β-Lactam Antibiotics by Penicillin-binding Protein 2a of Methicillin-resistant Staphylococcus aureus

Crystal structure of the carbapenemase OXA-24 reveals insights into the mechanism of carbapenem hydrolysis

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