The importance of the negative charge of <i>β</i>-lactam compounds in the interactions with active-site serine <scp>dd</scp>-peptidases and <i>β</i>-lactamases

Varetto, Louis; Meester, Fabien De; Monnaie, Didier; Marchand‐Brynaert, J.; Dive, Georges; Jacob, F; Frère, Jean‐Marie

doi:10.1042/bj2780801

Cited by 34 publications

(37 citation statements)

References 23 publications

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“…This neutral form would then be in place to act as a catalytic base, ultimately to the Tyr150 in deacylation but possibly also for acylation of Ser64 by a β-lactam substrate, as has been proposed by others. 10,26,38 This coordinate base hypothesis, though consistent with the ultrahigh-resolution structure on its own, is disfavored by previous biochemical experiments. Perhaps the strongest evidence against this hypothesis is the effect of the substitution Lys67→Arg, explored by Frère and colleagues.…”

Section: Discussionsupporting

confidence: 53%

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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Section: Discussionsupporting

confidence: 53%

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

et al. 2006

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“…26,33 The energetic contribution of the primary carboxylate site of -lactams has only begun to be investigated for AmpC, but on the basis of studies of class A -lactamases, this site may contribute 3-4 orders of magnitude in binding affinity. 24,44,45 The contribution of the carbonyl/hydroxyl binding sites is not possible to estimate due to the covalent mechanism of the -lactams and the inability to uncouple the contributions of the hydroxyls from those of the boron in the boronic acids. Nevertheless, these binding sites appear to be regions of favorable energetic complementarity to AmpC.…”

Section: Discussionmentioning

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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“…According to the results of a quantum mechanics calculation study by Vaterro et al, however, the orientation of the C4-carboxyl group differed from that of the C3-carboxyl group of penicillins. 35) Moreover, structural analysis of the cepharoridineacylated double mutant (Glu166Asp:Asn170Gln) showed that the C4-carboxyl group made a weak hydrogen bond with Ser130O g (The C4-carboxyl oxygen-Ser130O g distance was found to be 3.78 Å.). 36) From these findings, the C4-carboxyl group seems to be inappropriate for a proton abstractor from the hydroxyl group of Ser130.…”

Section: Deacylation Mechanism Of Class a B-lacta-masementioning

confidence: 99%

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Hata

Fujii

Tanaka

et al. 2006

Biological & Pharmaceutical Bulletin

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INTRODUCTIONb-Lactamases are produced by pathogenic bacteria and are enzymes that cause resistance to b-lactam antibiotics by hydrolyzing their b-lactam rings.1-3) b-Lactamases are classified into two major types on the basis of the main component of the active site: serine-b-lactamases and metallo-b-lactamases. 4,5) Serine-b-lactamases are further classified into three classes: classes A, C, and D; i.e., metallo-b-lactamase is classified into class B.As is well known, the catalytic mechanism of serine-b-lactamases involves acylation ( Fig. 1 (a) to (c) via (b)) and deacylation ( Fig. 1 (c) to (d)). The acylation mechanism has been examined in many experimental [6][7][8][9][10][11][12][13][14] and theoretical works, 11,[15][16][17][18][19][20][21][22] and acylation is common to serine-b-lactamase and penicillin-binding protein (PBP). Deacylation, on the other hand, is only seen in serine-b-lactamase. This means that the essence of antibiotic resistance by serine-blactamase arises from the deacylation reaction. Hence, an understanding of the reaction mechanism of deacylation is important for developing novel b-lactam antibiotics and potent b-lactamase inhibitors. We have investigated the deacylation mechanisms of serine-b-lactamases by using theoretical calculation. In this paper, the results of our recent computational analyses for all classes of serine-b-lactamases are presented. DEACYLATION MECHANISM OF CLASS A b-LACTA-MASEClass A b-lactamase is more frequently detected in a clinical setting than are other classes of b-lactamase. Since the study by Knox et al. suggested that the E166A mutant of blactamase from Bacillus licheniformis induced the accumulation of an acyl-enzyme intermediate, 23) the catalytic residue involved in the deacylation reaction has been presumed to be only Glu166. However, it is difficult to conclude that the deacylation reaction occurs only due to Glu166 because the distance between Ser70O g and Glu166O e is too far to interact (ca. 3.74 Å) according to the results of our molecular dynamics (MD) simulation of the structure of b-lactam antibioticsb-lactamase complex. 24) We have speculated that not only Glu166 but also Lys73 is involved in the deacylation reaction because Lys73N z is located between Ser70 and Glu166 and interacts with Ser70O g and Glu166O e , nearly forming hydrogen bonds (2.77 and 3.21 Å, respectively).24) Furthermore, a hydrogen bond network among Lys73N z , Ser130O g and the carboxyl group of the substrate (b-lactam antibiotics) should be taken into account to clearly explain the reaction mechanism. Ishiguro et al. also discussed the importance of the hydrogen bond network based on the results of their molecular mechanics calculations.15) Accordingly, we investigated the whole mechanism of the deacylation reaction by theoretical The substrate deacylation mechanisms of serine-b b-lactamases (classes A, C and D) were investigated by theoretical calculations. The deacylation of class A proceeds via four elementary reactions. The rate-determining process is the tetrahedra...

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The importance of the negative charge of β-lactam compounds in the interactions with active-site serine dd-peptidases and β-lactamases

Cited by 34 publications

References 23 publications

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

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

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

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