On the Origin of Bacterial Resistance to Penicillin: Comparison of a β-Lactamase and a Penicillin Target

Kelly, Judith A.; Dideberg, Otto; Charlier, Paulette; Wery, Jean-Pierre; Libert, Marie; Moews, Paul C.; Knox, James R.; Duez, Colette; Fraipont, Claudine; Joris, Bernard; Dusart, Jean; Frère, Jean‐Marie; Ghuysen, Jean‐Marie

doi:10.1126/science.3082007

Cited by 218 publications

(150 citation statements)

References 16 publications

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“…Ampicillin (4 mg) was dissolved in 55 p1 of a mixture of acetonitrile and 1 M NH4HC03 solution (9/1; v/v). The solution was supplemented with 1 p1 of 0.5% [14C]acetic anhydride in toluene (Amersham; 120 Cij mol) and maintained for 2 h at 20 "C. Non-radioactive acetic anhydride (20 PI) and acetonitrile (10 pl) were added and after 16 h at 20°C, the reaction mixture was evaporated to dryness and the residue dissolved in 2 ml 50 mM sodium phosphate pH 7.0. The N-acetylampicillin concentration (55 mM) was estimated by measuring both the absorbance of the solution at 230 nm ( E = 2400 M-cm-') and the rate with which the preparation inactivated the R61 DD-peptidase (using the nonradioactive N-acetylampicillin as control).…”

Section: Appendixmentioning

confidence: 99%

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

Varetto

Frère

Nguyen-Distèche

et al. 1987

European Journal of Biochemistry

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Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac,-~-Lys-~-Aia-~-Ala and enzyme inactivation by the p-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK z 9.5. It is proposed that protonation of a lysine &-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the 8-lactam molecules. Lowering the pH from 7 to 5 has no effect on the secondorder rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac,-~-Lys-~-Ala-~-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5 -7.0 in the hydrolysis pathway.Two families of bacterial enzymes specifically interact with p-lactam compounds. The p-lactamases, which hydrolyse the p-lactam ring, can play an important role in resistance. The DD-peptidases, which participate in the last stages of wall peptidoglycan metabolism, are the primary targets of these antibiotics [l, 21. Many of the /3-lactamases (classes A and C) and DD-peptidases characterized so far are serine enzymes and react with p-lactam compounds according to reaction (1).(1)where E = enzyme, D = p-lactam, k + and k , , = first-order rate constants, K = dissociation constant of E . D (Michaelis complex) and E -D* = serine ester-linked acyl enzyme [3]. Most often, the value of k , , is high or very high with the j-lactamases, and so low with the DD-peptidases that a slight stoichiometric excess of p-lactam compound causes longlasting immobilization of the protein at the level of the acyl enzyme and these latter enzymes behave as penicillin-binding and C, the active-site serine is followed by an Xaa-Xaa-Lys sequence (which suggests that this lysine probably plays an important role), little is known concerning the enzymes' functional groups involved in substrate binding and catalysis. where HY is H 2 0 or a suitable R-NH2 peptide or amino acid. The goal of the present work was to obtain further information on the possible functional groups involved in catalysis and penicillin binding by the Streptomyces R61 DD-peptidase. When this study was initiated, it was known [2] that at pH 7.0 and 37°C (unless otherwise stated) (a) the K , k + , and k+, values for the interaction with benzylpenicillin were 13 mM (25"C), 180s-I (25°C) and 1 . 4~1 0 -~s -' ; (b) the corresponding values for the interaction with ampicillin were 7.2 mM, 0.77 s-l and 1.4 x s-'; (c) the K,,, and k,,, values for the best substrate Ac,-~-Lys-~-Ala-~-Ala were about 10 mM and 50 S K I , respectively and (d) the activity of the enzyme decreased with increasing ionic strength [15]. MATERIALS AND...

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Section: Appendixmentioning

confidence: 99%

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

Varetto

Frère

Nguyen-Distèche

et al. 1987

European Journal of Biochemistry

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“…Previously, differential protein stability has been used to measure the thermodynamic complementarity of both ␤-lactam and non-␤-lactam ligands for ␤-lactamase enzymes (Rahil and Pratt 1994;Beadle et al 1999), which are structurally and functionally related to PBPs (Kelly et al 1986;Massova and Mobashery 1998;Fonze et al 1999;Davies et al 2001). Rather than measuring the equilibrium constant between bound and free ligand, this technique measures the equilibrium constant between folded and unfolded protein (Schellman 1976), in the presence and absence of a ligand.…”

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confidence: 99%

Interaction energies between β‐lactam antibiotics and E. coli penicillin‐binding protein 5 by reversible thermal denaturation

2001

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Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with ␤-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize ␤-lactam antibiotics, it is important to know their energies of interaction. Because ␤-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between ␤-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T m ) of 48.5°C and a van't Hoff enthalpy of unfolding (⌬H VH ) of 193 kcal/mole. The binding of the ␤-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with ⌬T m values as high as +4.6°C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k 2 /KЈ). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.Keywords: Penicillin-binding protein; PBP 5; ␤-lactam; ␤-lactamase; enzyme stability; denaturation Penicillin-binding proteins (PBPs) are involved in the biosynthesis of bacterial cell wall peptidoglycan. All bacteria analyzed to date have multiple PBPs, which function to crosslink the cell wall peptides (transpeptidation) and to remove the C terminal D-alanine residue from cell wall peptides (carboxypeptidation). PBPs are the targets of ␤-lactam antibiotics, which react with these enzymes to form stable covalent complexes, leading to inactivation of the PBP and ultimately to cell death (Tipper and Strominger 1965;Wise and Park 1965;Ghuysen and Dive 1994). A broad range of ␤-lactam antibiotics, including penicillins, cephalosporins, monobactams, and carbapenems, are used clinically, and seemingly subtle structural differences have dramatic effects on their biological activity (Neuhaus and Georgopapadakou 1992).Despite much interest in understanding the molecular interactions of ␤-lactams with PBPs, their interaction energies have been difficult to determine. The noncovalent first encounter complex between the antibiotic and enzyme proceeds quickly to an acyl-enzyme adduct by attack of the

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“…The alignment ( Figure 2) indicated that EstG34 contained the S-X-X-K consensus sequence (S-M-T-K 75 ). This S-X-X-K motif is conserved within most of the β-lactamase superfamily proteins, which includes penicillin binding proteins (PBPs), DD-peptidases and other family VIII carboxylesterases [39,40,41]. However, two other highly conserved class C β-lactamase motifs (Y-A-N) and (K-T/S-G) [42] were not found in EstG34 (Figure 2).…”

Section: Resultsmentioning

confidence: 91%

“…Interestingly, detailed selectivity assays conducted for Est22 [49] and EstB [40] demonstrated that the enzymes selectively hydrolysed the ester bond of a number of cephalosporin-based substrates, while leaving the amide bond of the β-lactam ring intact. This clearly differentiates them from bona fide β-lactamases.…”

Section: Discussionmentioning

confidence: 99%