Crystal Structures of Substrate and Inhibitor Complexes with AmpC β-Lactamase:  Possible Implications for Substrate-Assisted Catalysis

Patera, A.; Blaszczak, Larry C.; Shoichet, Brian K.

doi:10.1021/ja001676x

Cited by 92 publications

(192 citation statements)

References 52 publications

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“…these residues, as observed in the complexes of AmpC with 6, 8, and 12 20,24,26 (Table 1; Figure 3C). In the complexes of AmpC with the -lactams 10 and 13, the carbacephem and oxacephem rings, respectively, interact with the leucines.…”

Section: Resultssupporting

confidence: 65%

“…In this structure, the carboxylate also hydrogen bonds with Thr316. 20 In the AmpC/13 (moxalactam) complex, the C4 carboxylate hydrogen bonds directly with Asn289 and Asn346 and interacts, via water molecules, with Asn343 and Arg349. 20 None of the computational programs were used to predict carboxylate binding sites on AmpC.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the structure of AmpC has been determined in complex with inhibitors such as -lactams, 4,[20][21][22] transition state analogues, 23,24 and arylboronic acids, 6,25,26 the leads for which were known before the first structures were determined. 27 There are now 18 inhibitor complex structures with class C -lactamases that have been published.…”

Section: Introductionmentioning

confidence: 99%

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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: Resultssupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2002

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“…21 The proton on the Tyr150 Oη is collinear with the position of the lactam nitrogen on the other side of the boronic O2 atom and 2.63 Å away from the O2 atom. The tyrosine proton is thus well placed to stabilize developing negative charge on the deacylating water, while the substrate nitrogen acts as a base, transiently accepting the proton from the other side.…”

Section: Discussionmentioning

confidence: 99%

“…7,21,29 In the deacylation transition state, analogous to this structure, it has been suggested that Lys67 can cooperate with Tyr150, either stabilizing a tyrosinate base or accepting a proton from the hydrolytic water by way of the tyrosine. 24,25 In either scenario, Lys67 might be expected to hydrogen bond with Tyr150.…”

Section: Lys67mentioning

confidence: 98%

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

et al. 2006

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Abstractβ-Lactamases confer bacterial resistance to β-lactam antibiotics, such as penicillins. The characteristic class C β-lactamase AmpC catalyzes the reaction with several key residues including Ser64, Tyr150, and Lys67. Here, we describe a 1.07 Å X-ray crystallographic structure of AmpC β-lactamase in complex with a boronic acid deacylation transition-state analogue. The high quality of the electron density map allows the determination of many proton positions. The proton on the Tyr150 hydroxyl group is clearly visible and is donated to the boronic oxygen mimicking the deacylation water. Meanwhile, Lys67 hydrogen bonds with Ser64Oγ, Asn152Oδ1, and the backbone oxygen of Ala220. This suggests that this residue is positively charged and has relinquished the hydrogen bond with Tyr150 observed in acyl-enzyme complex structures. Together with previous biochemical and NMR studies, these observations indicate that Tyr150 is protonated throughout the reaction coordinate, disfavoring mechanisms that involve a stable tyrosinate as the general base for deacylation. Rather, the hydroxyl of Tyr150 appears to be well positioned to electrostatically stabilize the negative charge buildup in the tetrahedral high-energy intermediate. This structure, in itself, appears consistent with a mechanism involving either Tyr150 acting as a transient catalytic base in conjunction with a neutral Lys67 or the lactam nitrogen as the general base. Whereas mutagenesis studies suggest that Lys67 may be replaced by an arginine, disfavoring the conjugate base mechanism, distinguishing between these two hypotheses may ultimately depend on direct determination of the pK a of Lys67 along the reaction coordinate.

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Antibiotic Resistance

Golemi-Kotra

Mobashery

2002

Kirk-Othmer Encyclopedia of Chemical Technology

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Resistance to antibiotics has created a state of crisis in clinics for treatment of infections. Historically, the majority of known classes of antibiotics have come from natural sources, examples of which are macrolides,β‐lactams, and aminoglycosides, among others. They target vital bacterial biochemical events, such as cell wall synthesis, protein synthesis, and DNA replication. A description of these process is provided herein. Furthermore, resistance to known antibiotics has emerged by a multitude of mechanisms, a subject that has been elaborated in this article in detail.

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Crystal Structures of Substrate and Inhibitor Complexes with AmpC β-Lactamase: Possible Implications for Substrate-Assisted Catalysis

Cited by 92 publications

References 52 publications

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

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

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

Antibiotic Resistance

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