Abstract
Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.
Highlights
The structures of several MBLs have been solved by x-ray diffraction and reveal two potential zinc ion binding sites at the active site (6 –16)
Assignment of the 113Cd Spectrum of Cadmium-substituted B. cereus MBL—As described earlier [24, 25], the BcII enzyme with two cadmium ions bound gives a 113Cd NMR spectrum containing two distinct resonances at ϳ140 and ϳ260 ppm (Fig. 1A)
The lower field 113Cd resonance gives rise to one imidazole 1H resonance, at 7.0 ppm, and one 1H resonance in the aliphatic region, at 1.6 ppm; this 113Cd signal can be assigned to the metal in site 2, the imidazole resonance being that of His210 (BBL263) (consistent with the results of studies of the zinc/cadmium hybrid enzyme3 and the aliphatic resonance being that of one or both of the -protons of Cys168 (BBL221)
Summary
Materials—15N-Labeled protein was expressed from plasmid pET/ BCII in E. coli BL21(DE3). The apoenzyme was added, giving a final enzyme concentration of 0.5 mM in the PAC samples. PAC Data Analysis—The perturbation function, G2(t), of the angular correlation pattern is derived from the measurements as described previously (29 –32). This function depends on the local electric quadrupole interaction at the site of the cadmium nucleus and contains the structural information about the co-ordination geometry that can be deduced from a PAC experiment. In the case of experiments with S-thiomandelate, it was possible (judged by acceptable values for the reduced 2 function) to fit the spectra obtained at different [cadmium]/. Thiomandelate was docked into structures where Lys171 is in an extended conformation (see “Discussion”); the inhibitors were removed from the published structures, and the protein and ligand were parameterized as described by Hanessian et al [37]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
