Metallo-P-lactamases

Because over half of aU the commercially available antibiotics are 3-lactams, and in recent years bacterial resistance to these molecules has become so prevalent (to the point that some infections can no longer be treated), metaUo-jJ-lactamases are not only important from the organometallic standpoint but they are enzymes central to human health [229]. Most of the p-lactamases identified to date belong to the A, C and D serine hydrolase classes [230-233]. Class B enzymes, on the other hand, require divalent (Zn, Co, Cd or Mn) metal ions for catalytic activity [234, 235]. Because only 10% of the 200-odd P-lactamases identified so far are metaUoenzymes little attention has been given to these proteins as potential threats to antibiotic therapy [236,237]. However, metallo-p-lactamases are able to hydrolyze compoimds that are resistant to serine-p-lactamases and they show little or no susceptibility to traditional p-lactamase inhibitors [238, 239]. This has resulted in ineffective use of the most available antibiotics in bacteria capable of producing metaUo-P-lactamases [240, 241]. 

In general these enzymes catalyze the hydrolytic opening of the p-lactam ring according to the reaction depicted in Fig. 11.26. The hydrolytic product, unlikely the intact P-lactam, is not able to interfere with bacterial cell wall synthesis, the key effect of the antibiotic [229]. The crystal structures of several metaUo-p-lacta-mases have shown that the active site can have one or two Zn(ll) ions (Znl and Zn2) then separated by 3.5 A [242-247]. In these structures Znl is tetrahedraUy coordinated to three His and one water molecule. When present, Zn2 is either penta-coordinated to Cys, His and Asp residues and two water molecules, or to two His, one Asp and two water molecules, in a trigonal bipyramid. In the structure where the active site is composed of a binuclear Zn center, a H20/0H bridges the two metals. As in other binuclear centers with bridging solvent molecules, its activation provides the OH nucleophile for the reaction [248]. It should be pointed out that not all P-lactam antibiotics bind to the active site in the same way so mechanisms may differ from molecule to molecule. 

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P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

Class B Metallo p-lactamases L1 enzyme of S. maltophilia enzyme of Aeromonas spp. CcrA enzyme found in 1-3% of 8. fragilis isolates. All enzymes are carbapenemases IMP, VIM and SPM type carbapenemases... 

Scheme 7.3 (a) Anchor fragments equipped with two thiol moieties (F-J). (b) Thiol partner fragments (4-22). (c) DCC ntilizing thiol-disulfide exchange and targeting metallo-P-lactamase (Bell). 

Felici A, Perilli M, Segatore B, Franceschini N, Setacci D, Oratore A, Stefani S, Galleni M, Amicosante G (1995) Interactions of biapenem with active-site serine and metallo-P-lactamases. Antimicrob Agents Chemother 39 1300-1305. 

G. G. Hammond, Chem. Biol, 5, 185 (1998). Antibiotic Sensitization Using Biphenyl Tetrazoles as Potent Inhibitors of Bacteroides fragilis Metallo-P-lactamase. 

Fig. 5.16. Strategy for the selection of a metallo-P-lactamase (Bla) displayed on phage [69], The phage-enzyme is inactivated by extracting the metallic cofactor and captured with an immobilised penicillin substrate. Addition of the metallic cofactor results in the catalytic elution of the phage-enzyme.

D. E. Vanderwall, K. A. Cleary, S. K. Grant, J. K. Wu, J. W. Kozarich, D. L. Pompliano, and G. G. Hammond, Chem. Biol., 5, 185 (1998). Antibiotic Sensltatlon Using Biphenyl Tetrazoles as Potent Inhibitors of Bacteroides fragilis Metallo-P-Lactamase. 

Many enzymes contain two or more metal ions in the active site, exploiting collaboration among the metal centers in the catalytic action. Examples of multinuclear metalloenzymes catalyzing hydrolysis of acyl derivatives and related compounds are methionine aminopeptidase (4), metallo-p-lactamase... 

Metallo-p-lactamases that are poorly inhibited by all classical p-lactamase inhibitors... 

Three of the four classes of (3-lactamases, A, C, and D are serine nucleophile-based enzymes, while the fourth, class B, contains zinc metallo-p-lactamases. Among the serine (3-lactamases, classes A and C are currently the most intensely studied. However, extended spectrum class D enzymes have, of late, been growing in clinical importance [309 314]. 

Metallo-p-lactamases as possible weaponry for antibiotic resistance in bacteria 06ACR72I. 

Kaminskaia, N.V. Spingler. B. Lippard, S.J. Hydrolysis of p-lactam antibiotics catalyzed by dinuclcar zinc(Il) complexes Functional mimics of metallo-P-lactamases. J. Am. Chem. Soc. 2000, 122 (27). 6411-6422. 

Antibacterial activities of novel active pharmaceutical ingredient ionic liquids based on ampicillin 31 have been evaluated (14MI4301). Structural and mechanistic insights into New Delhi metallo-P-lactamase catalyzed hydrolysis of cephalosporins have been reported (14JA14694). The hydrolysis of the antibiotic meropenem 32 by Escherichia coli cells carrying the gene for New Delhi metallo-P-lactamase, which confers antibiotic resistance, has been monitored by NMR spectroscopy in real time (14AGE2130). An... 

Figure 5.19 Thiol (A/-benzoyl-D

Metallo-P- lactamases 3a B Metallo-P-lactamases (usually Ztf+ -dependent)... 

Metallo-P-lactamases [20] Zn(II)-Zn(II) Hydrolysis of P-lactam substrates (antibiotics) Study potential inhibitors to fight antibiotic resistance... 

Metallo-P-lactamase-like Activity Assays were conducted in the same aqueous multi-component buffer as mentioned above with the ionic strength controlled by 250 mM LiC104. Assays were carried out at 37 °C in 50 50 acetonitrileibufifer, with nitrocefin as substrate initially dissolved in acetonitrile (10 mM) and the complex dissolved in acetonitrile water (1 mM). Assays conducted to investigate pH dependence were 5 pM in complex and 50 pM in nitrocefin. [Substrate]... 

Fig. 5.3 The active site of the metallo-P-lactamase from B. fragilis and the proposed mechanism of cephalosporin hydrolysis [29]...

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