Inhibition of Metallo-b-lactamase

Spectroscopic Studies of the Bacillus cereus 5/B/6 Metallo-  -lactamase Cynthe L. Sims 1 , Sung-Kun Kim 2 , Mitchel L. Cottenoir 1 , Kyu-Me Kim 1 , Dustin Whitson 1 , William K. Myers 3 , Andrew J. Fisher 4 , David L Tierney 3 , and Robert Shaw 1 . (1) Department of Chemistry and Biochemistry, Texas Tech University, PO Box 41061, Lubbock, TX 79409-1061, (2) Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76706, (3) Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, (4) Department of Chemistry, UC Davis, Davis, CA 95616 Abstract ß-Lactam antibiotics are the most important drugs used to fight bacterial infection. The overuse and misuse of ß-lactam antibiotics in the clinical and agricultural fields has caused the evolution of resistance pathways, rendering pathogenic bacteria resistant to treatment. The major source of resistance is the production of enzymes called ß-lactamases capable of catalyzing hydrolysis of the ß-lactam. These ß-lactamases can be divided into several classes, yet it is the metallo-ß-lactamases, with either one or two zinc ions at the active site that have recently become a major threat. Currently, there are no clinically useful inhibitors for these enzymes. The metallo-ß-lactamase from Bacillus cereus 5/B/6 is capable of catalyzing the hydrolysis of a variety of cephalosporin antibiotics including cephalosporin C, cephalexin, and cefuroxime. We have previously reported the discovery of single stranded DNA, double stranded DNA and RNA aptamers capable of inhibiting this enzyme in the nanomolar range. Kinetic studies revealed a non-competitive mechanism of inhibition, suggesting a possible interaction of the aptamer with the metal ions at the active site, thereby inhibiting catalysis. Reconstitution of this enzyme with cobalt leads to the same non-competitive inhibition pattern as obtained with the native zinc form. Results from a variety of electronic and EPR spectroscopy, XAS and X- ray diffraction experiments aimed at investigating the chemical mechanism of inhibition will be discussed. Introduction Bacterial resistance to antibiotics has recently become a major medical crisis. Antibiotics that were once clinically useful in the treatment of infectious diseases are now no longer effective at killing resistant pathogens. The most problematic resistance mechanism originates from enzymes called  -lactamases that catalyze the hydrolysis of the amide bond in  -lactam antibiotics. Furthermore, it is the metallo-  -lactamases with one or two zinc ions at the active site that are medically troubling. Easily spread from one bacterial species to another through plasmids, the metallo-  -lactamases are capable of easily catalyzing the hydrolysis of all medically important  -lactam antibiotics including penicillins, and cephalosporins. There are no clinically useful inhibitors of this class of  -lactamases. The metallo-  -lactamase from B. cereus 5/B/6 has two Zn 2+ ions bound at the active site. The Zn1 site’s ligand set is three histidine residues, and the Zn2 site’s ligands are aspartate, cysteine, and histidine. A water is a bridging ligand between the two sites (1, 2). A hydroxide ion at the Zn1 site participates in base-catalyzed hydrolysis of the  -lactam; yet the contribution of Zn2 to catalysis is still being investigated. A combinatorial method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to derive an oligonucleotide inhibitor of the metallo-  -lactamase from B. cereus 5/B/6 (3, 4). Through this approach, we were able to derive a 30-mer capable of inhibiting enzymatic activity. Secondary structure analysis of the 30-mer by the Mfold program (4, 5) predicted a 10 residue stem-loop segment. Kinetic studies with the 10-mer revealed a noncompetitive mechanism of inhibition, suggesting a possible interaction of the aptamer with the metal ions at the active site. Spectroscopic studies allude to the possibility that the site of interaction of this inhibitor is with the Zn2 site’s metal ion; therefore giving support to the argument that the Zn2 site is also important to catalysis. Figure1. EXAFS fit of the mono- Zn B. cereus 5/B/6 metallo-  -lactamase inhibitor complex. Figure 2. EXAFS spectra of the di- Zn B. cereus 5/B/6 metallo-  -lactamase. The black line is the spectrum of the inhibited enzyme, the red line is that of the inhibited enzyme. Figure 3. Visible electronic spectra overlay of the Co(II) reconstituted B. cereus 5/B/6 metallo-  -lactamase. The blue line is the spectrum of the uninhibited enzyme, and the red line is the spectrum of the inhibited species. Results and Discussion X-ray absorption spectroscopy of the mono-Zn enzyme (Fig. 1) essentially shows no obvious change in the ligand environment of the Zn1 site upon addition of the inhibitor. Fitting routines gave the best results when 3 histidines and 1 oxygen were used; this is the same ligand set as the uninhibited enzyme. However, when the spectrum of the di-Zn enzyme (Fig. 2) is compared with that of the inhibitor complex, a perturbation is noted in the primary coordination sphere. This perturbation could arise from either a change in the ligand environment cause by binding of the inhibitor near the metal ligands or by direct coordination of a metal by atoms from the inhibitor. In either case, these data suggest inhibition is due to a perturbation at the Zn2 site. Visible spectra of the uninhibited and inhibited Co 2+ -reconstituted enzymes (Fig. 3) are consistent with a perturbation of the coordination of the metal ion at the active site upon binding of the inhibitor. Notably, the ligand to metal charge transfer band at 342 nm undergoes a blue shift to 339nm and an increase in absorbance; the other bands remain essentially the same. Previous studies on substrate binding show a large increase in this ligand to metal charge transfer band as a result of cysteine 168 moving closer to the metal ion in the Zn2 site. (6, 7, 8). Given the evidence that these experiments present it can be concluded that the inhibitor interacts with the enzyme in a way that perturbs the primary coordination sphere of the Zn2 site and it is this interaction that is responsible for inhibition of catalysis. These observation lends support to the idea that the Zn2 site is required for catalysis (9). We expect that further EXAFS experiments may allow us to acquire higher resolution data so that fits can be made and conclusions can be drawn as to how the inhibitor specifically interacts with the metal ion. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Acknowledgements Travel support (C.L.S.) was provided by the Texas Tech University Graduate School.

Inhibition of Metallo-b-lactamase

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Inhibition of Metallo-b-lactamase