Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.     

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)     

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC)

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.     

Mechanistic studies on the mononuclear ZnII-containing metallo-beta-lactamase ImiS from Aeromonas sobria

In an effort to understand the reaction mechanism of a B2 metallo-beta-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s(-)(1) and a slower rate of fluorescence regain of 98 s(-)(1). Stopped-flow UV-vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s(-)(1). These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-beta-lactamases and demonstrate that the mono- and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.     

Characterization of an inhibitory metal binding site in carboxypeptidase A

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases

Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.     

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Extended X-ray absorption fine structure studies of the metallo-β-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn₁ site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal–metal interaction at 3.42 Å. Reaction with the β-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn–Zn interaction to 3.62 Å.     

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase

The metal content of bovine NADH-Q oxidoreductase determined by inductively-coupled plasma atomic-emission spectroscopy reveals the presence of about one atom of zinc per molecule of flavin mononucleotide. We applied Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) to investigate the local structure of the bound zinc ion and to identify the nature of the coordinating residues. The EXAFS spectrum is consistent with a structured zinc binding site. By combining information from first-shell analysis and from metalloprotein data bases putative binding clusters have been built and fitted to the experimental spectrum using ab initio simulations. The best fitting binding cluster is formed by two histidine and two cysteine residues arranged in a tetrahedral geometry.     

The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives

The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe(3+) and Zn(2+) derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin.The binding of both metals is accompanied by small changes in the amino acid ligand positions. Mean Fe(A)(3+)-Fe(B)(3+) and Zn(A)(2+)-Zn(B)(2+) distances are 3.24 A and 3.43 A, respectively. In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation. The Fe(3+) derivative alone shows a third metal site (Fe( C)( 3+)) joined to Fe(B)(3+) by a long anti-anti bidentate bridge through Glu130 (mean Fe(B)(3+)-Fe(C)(3+) distance 5.79 A). The third metal site is unique to the non-haem bacterial ferritins.The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water. Models representing the possible binding mode of dioxygen to the dinuclear Fe(3+) pair suggest that a gauche micro-1,2 mode may be preferred stereochemically.Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles. The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s. deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %). Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions.     

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site

We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (micro-aqua)(micro-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn(1) ion at the enzyme C-terminus, following the binding of AMP at the Zn(2) ion located at N-terminus of the enzyme. Thus, the two Zn ions in the AMPD metallocenter operate together as a single catalytic unit, but have independent function, one of them (Zn(1)) acting to polarize the nucleophile water molecule, whilst the other (Zn(2)) acts transiently as a receptor for an activating substrate molecule. The addition of fluoride to AMPD also abolishes the cooperative behaviour induced in the enzyme by the inhibitory effect of ATP at acidic pH that probably resides in the competition with the substrate for an adenine nucleotide specific regulatory site located in the Zn(2) ion binding region and which is responsible for the positive homotropic cooperativity behaviour of AMPD.     

Flexible metal binding of the metallo-beta-lactamase domain: glyoxalase II incorporates iron, manganese, and zinc in vivo

Glyoxalase II belongs to the metallo-beta-lactamase superfamily of proteins, possessing the characteristic dinuclear active site. Within this protein family, glyoxalase II from Arabidopsis thaliana is the first member to be isolated with significant amounts of iron, manganese, and zinc when being recombinantly produced in Escherichia coli. Enzyme preparations with different ratios of these three metals all yield k(cat)/K(M) values in the range of 1.5-1.9 s(-1) microM(-1) with the substrate S-d-lactoylglutathione. X-ray absorption spectroscopy reveals binding of all three metals to the dinuclear active site with 5-6-fold coordination consisting of 2.5 +/- 0.5 histidine and 2.5 +/- 0.5 oxygen ligands. This model does not distinguish site-specific or distributed binding. The metal-metal distance is determined to be 3.18 +/- 0.06 A. Electron paramagnetic resonance spectroscopy gives evidence for several different types of dimetal sites, including spin-coupled Fe(III)Fe(II), Fe(III)Zn(II), and Mn(II)Mn(II) centers. The metal-ligand distances measured by X-ray absorption spectroscopy vary depending on the metal type and comply with their element-specific, characteristic values. This reflects a high degree of structural flexibility within the glyoxalase II dinuclear active site, which is considered as the structural basis for its broad metal selectivity.     

In vivo folding of recombinant metallo-beta-lactamase L1 requires the presence of Zn(II)

Metallo-beta-lactamase L1, secreted by pathogenic Stenotrophomonas maltophilia, is a dinuclear Zn(II)-containing enzyme that hydrolyzes almost all known penicillins, cephalosporins, and carbapenems. The presence of Zn(II) ions in both metal binding sites is essential for full enzymatic activity; however, the mechanism of physiological metal incorporation is unknown. To probe metal incorporation, L1 was over-expressed in minimal media with (mmL1+Zn) and without (mmL1-Zn) Zn(II) added to the media, and the resulting proteins were purified and characterized. The mmL1+Zn sample was bound by a Q-Sepharose column, exhibited steady-state kinetic properties, bound Zn(II), existed as a tetramer, and yielded fluorescence emission and CD spectra similar to L1 overexpressed in rich media. On the other hand, the mmL1-Zn sample did not bind to a Q-Sepharose column, and gel filtration studies demonstrated that this protein was monomeric. The mmL1-Zn sample exhibited a lower kcat value, bound less Zn(II), and yielded fluorescence emission and CD spectra consistent with this enzyme being folded improperly. Taken together, these data demonstrate that the proper folding of L1 requires the presence of Zn(II) and suggest that in vitro, thermodynamic metal binding studies do not accurately reflect physiological metal incorporation into L1.     

Metal binding in amyloid β-peptides shows intra- and inter-peptide coordination modes

X-ray absorption spectroscopy data show different metal binding site structures in beta-amyloid peptides according to whether they are complexed with Cu(2+) or Zn(2+) ions. While the geometry around copper is stably consistent with an intra-peptide binding with three metal-coordinated Histidine residues, the zinc coordination mode depends on specific solution conditions. In particular, different sample preparations are seen to lead to different geometries around the absorber that are compatible with either an intra- or an inter-peptide coordination mode. This result reinforces the hypothesis that assigns different physiological roles to the two metals, with zinc favoring peptide aggregation and, as a consequence, plaque formation.     

Unraveling the substrate-metal binding site of ferrochelatase: an X-ray absorption spectroscopic study

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)(3)(S/Cl)(1) coordination for the yeast and Zn(N/O)(2)(S/Cl)(2) coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co(2+) coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co(2+), Fe(2+) bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.     

Structural analysis of ABC-family periplasmic zinc binding protein provides new insights into mechanism of ligand uptake and release

ATP-binding cassette superfamily of periplasmic metal transporters are known to be vital for maintaining ion homeostasis in several pathogenic and non-pathogenic bacteria. We have determined crystal structure of the high-affinity zinc transporter ZnuA from Escherichia coli at 1.8 A resolution. This structure represents the first native (non-recombinant) protein structure of a periplasmic metal binding protein. ZnuA reveals numerous conformational features, which occur either in Zn(2+) or in Mn(2+) transporters, and presents a unique conformational state. A comprehensive comparison of ZnuA with other periplasmic ligand binding protein structures suggests vital mechanistic differences between bound and release states of metal transporters. The key new attributes in ZnuA include a C-domain disulfide bond, an extra alpha-helix proximal to the highly charged metal chelating mobile loop region, alternate conformations of secondary shell stabilizing residues at the metal binding site, and domain movements potentially controlled by salt bridges. Based on in-depth structural analyses of five metal binding transporters, we present here a mechanistic model termed as "partial domain slippage" for binding and release of Zn(2+).     

Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The biochemical properties of recombinant ImiS were compared with those of native ImiS. Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures. Gel filtration chromatography revealed that both enzymes exist as monomers in solution. MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca. 0.7 mol of Zn(II). Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc. Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme. This over-expression protocol now allows for detailed spectroscopic and mechanistic studies on ImiS as well as site-directed mutants of ImiS to be prepared for future structure/function studies.     

Azolylthioacetamides as a potent scaffold for the development of metallo-β-lactamase inhibitors

In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl and benzioxazolyl substituted 1–19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC₅₀ value of 15?nM. The nitrobenzimidazolyl substituted 20–28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC₅₀ value of 170?nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC₅₀ values. These inhibitors resulted in a 2–4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.