Untitled

Baleja, James D.; Thanabal, V.; Wagner, Gerhard

doi:10.1023/a:1018332327565

Cited by 17 publications

(13 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 3 J H-Cd scalar coupling constants to the inhibitor C␣ proton are notably different between the complexes with R-thiomandelate (15 and 31 Hz) and S-thiomandelate (Ͻ15 Hz). The magnitudes will depend on the orientation of the inhibitor in the complex, through the dihedral angle about the thiomandelate C␣ϪS bond, but also on the electron distribution on the metals and their ligands, and clear-cut relationships to conformation can rarely be discerned (25,39). We earlier noted that the changes in backbone amide 1 H and 15 N chemical shifts on inhibitor binding to the zinc enzyme are generally very similar for the two isomers (23), suggesting that the structural differences between the two complexes are local ones.…”

Section: Discussionmentioning

confidence: 99%

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Damblon

Jensen²,

Ababou

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lactamases, enzymes that mediate resistance to ␤-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-␤-lactamase. The 113 Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113 Cd-edited 1 H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113 Cd 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 H␣ 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.␤-Lactamases inactivate ␤-lactam antibiotics by catalyzing the hydrolysis of their endocyclic amide bond and play a major role in the emergence of strains of pathogenic bacteria resistant to these important antibiotics (1). The ␤-lactamases that contain a nucleophilic serine side chain as a key component of their active site (classes A, C, and D) have been extensively studied due to their established clinical importance. The class B ␤-lactamases or metallo-␤-lactamases (MBLs) 1 are metalloproteins that require one or two zinc ion(s) for their activity (2) and are less well understood. The first of these enzymes to be discovered was produced by an innocuous strain of Bacillus cereus (3), but in the last 20 years, MBL-mediated resistance has appeared in several pathogenic strains and is being rapidly spread by horizontal transfer, involving both plasmid-and integron-borne genetic elements (4, 5).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). The zinc ligands are not fully conserved between the different subclasses of MBL. In the subclass B1 enzymes, such as the B. cereus enzyme BcII, which is the subject of the present work, the zinc in site 1 is coordinated by the imidazole rings of three histidine residues (86, 88, and 149 in the B. cereus enzyme or 116, 118, and 196 using the class B ␤-lactamase (B...

show abstract

Section: Discussionmentioning

confidence: 99%

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Damblon

Jensen²,

Ababou

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…GAL4 [57], was unprecedented for any MT species so far. In continuation of the nomenclature used for the two mammalian MT domains, the N-terminal domain of wheat E c -1 was denominated as -domain.…”

Section: >> Insertmentioning

confidence: 99%

Structural features specific to plant metallothioneins

Freisinger

2011

J Biol Inorg Chem

106

View full text Add to dashboard Cite

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn(II), Cd(II), and Cu(I). The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E(c)-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal-thiolate cluster structures. Very peculiar and not well understood is the presence of rather long Cys-free amino acid linkers between the Cys-rich regions. In light of the distinct differences in sequence to MTs from other families it seems obvious to assume that these differences will be also manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E c -1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family other sources of information are of high demand.In this review the focus is thus set on any structural features known, deduced or assumed for the plant MT proteins. This includes the determination of secondary structural elements by CD, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, as well as the evaluation of the spatial arrangement of the sequence separated Cys-rich regions with the aid of e.g. limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion-to-Cys ratios are important for predicting and

show abstract

“…21 Similar difficulties have been reported in the bi-metal zinc cadmium-substituted DNA-binding domain of GAL4 and presumably arise from the complexity of the electronic structure of the metal. 22 However, for the 1 J N-Cd values, there are indications of a possible relationship to the structure around the cadmium center.…”

mentioning

confidence: 99%

¹H−¹⁵N HMQC for the Identification of Metal-Bound Histidines in¹¹³Cd-SubstitutedBacillus cereusZinc β-Lactamase

et al. 1999

View full text Add to dashboard Cite

Cadmium is often used as a useful NMR 1 probe to study zincor calcium-containing proteins; 2 we present here a method for identification of cadmium-coordinated histidine via 1 H-15 N HMQC, which does not require any Cd excitation pulse. The cross-peaks show an E-COSY 3 type of pattern which allows the measurement of the 3 J H-Cd and 1 J N-Cd coupling constants.In many metalloproteins, notably those containing zinc or calcium, the natural metal is not conveniently detectable by NMR. To overcome this problem, substitution of the metal by cadmium has often been used. In particular, cadmium-113 NMR has often been used to study zinc-containing proteins; 2 the 113 Cd chemical shift is very sensitive to the nature, number, and geometric arrangement of the ligands within the coordination sphere. In many cases, the substitution of Zn 2+ by Cd 2+ has been shown to have only a modest effect on the catalytic activity of metalloenzymes. 4 113 Cd resonances are commonly detected by direct observation ( 113 Cd has spin 1 / 2 and a sensitivity 63% that of 13 C) or by inverse detection of 113 Cd scalar-coupled to 1 H. Cysteine, methionine, and histidine residues have been successfully identified as the coordinating ligands using 1 H-113 Cd HMQC, 113 Cdedited 1 H-1 H COSY or 1 H-113 Cd heteroTOCSY experiments. [5][6][7][8] These inverse experiments require a time-delay for transfer of magnetization between 1 H and 113 Cd spin, and also a knowledge of the 113 Cd chemical shift, in view of the very large chemical shift range of 113 Cd, which can also make direct detection difficult. These prerequisites make the experiments difficult in many cases.It would therefore be desirable to have a method by which the ligands of 113 Cd can be identified which does not rely on the polarization transfer between 1 H and 113 Cd spins. We describe here a method for identifying cadmium-coordinated histidines using a two-dimensional 1 H-15 N HMQC experiment which does not require Cd excitation pulses and which permits the identification of the histidine imidazole proton and nitrogen resonances of 113 Cd-bound imidazoles and the determination of the 3 J H-Cd and 1 J N-Cd coupling constants.We demonstrate the method using the zinc -lactamase from Bacillus cereus strain 569/H/9 (BCII). Zinc -lactamases 9 in pathogenic bacteria, which can be plasmid-encoded, confer resistance toward all the -lactam antibiotics and represent a real threat to antibiotic therapy because no inhibitors are presently available for clinical purposes. 10 The crystal structure 11 of -lactamase BCII shows two zinc cations in the active site, one (site I) coordinated by three histidines and one water molecule, and the other (site II) coordinated by a histidine, a cysteine, an aspartate, and an unknown molecule, likely to be a carbonate ion. These two zinc ions can be replaced by 113 Cd with minimal change in the kinetic parameters of the enzyme. The 113 Cd spectrum of the enzyme showed two signals, at 143 and 266 ppm relative to 0.1 M Cd-(ClO 4 ) 2 ; comparison with previously rep...

show abstract

Untitled

Cited by 17 publications

References 15 publications

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Structural features specific to plant metallothioneins

¹H−¹⁵N HMQC for the Identification of Metal-Bound Histidines in¹¹³Cd-SubstitutedBacillus cereusZinc β-Lactamase

Contact Info

Product

Resources

About

Untitled

Cited by 17 publications

References 15 publications

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Structural features specific to plant metallothioneins

1H−15N HMQC for the Identification of Metal-Bound Histidines in113Cd-SubstitutedBacillus cereusZinc β-Lactamase

Contact Info

Product

Resources

About

¹H−¹⁵N HMQC for the Identification of Metal-Bound Histidines in¹¹³Cd-SubstitutedBacillus cereusZinc β-Lactamase