RESUMEN
Cobalt and zinc binding by the subclass B1 metallo-ß-lactamase BcII from Bacillus cereus is examined by X-ray absorption spectroscopy, at various levels of metal loading. The data show that a significant amount of the dinuclear enzyme is formed, even at substoichiometric levels of metal loading, whether the added metal is Zn(II) or Co(II). Increasing metal addition, from 0.5 to 1.0 to 2.0eq/mol of enzyme, are shown to result in a more ordered active site. While Zn(II) appears to show no preference for the Zn(1) (3H) or Zn(2) (DCH) sites, the extended X-ray absorption fine structure (EXAFS) suggests that Co(II) shows a slight preference for the DCH site at low levels of added Co(II). The results are discussed in the context of similar metal-binding studies of other B1 metallo-ß-lactamases.
Asunto(s)
Bacillus cereus/enzimología , Proteínas Bacterianas/metabolismo , Metaloproteínas/metabolismo , Metales/metabolismo , Espectroscopía de Absorción de Rayos X/métodos , beta-Lactamasas/metabolismo , Algoritmos , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Cobalto/química , Cobalto/metabolismo , Cobalto/farmacología , Relación Dosis-Respuesta a Droga , Cinética , Metaloproteínas/química , Metales/química , Metales/farmacología , Modelos Moleculares , Estructura Terciaria de Proteína/efectos de los fármacos , Zinc/química , Zinc/metabolismo , Zinc/farmacología , beta-Lactamasas/químicaRESUMEN
RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Proteínas Ribosómicas/genética , Zinc/farmacología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Histidina/química , Histidina/genética , Histidina/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Factores de Tiempo , Espectroscopía de Absorción de Rayos X/métodos , Zinc/metabolismoRESUMEN
In an effort to probe for metal binding to metallo-ß-lactamase (MßL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.
Asunto(s)
Serratia marcescens/enzimología , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Cationes Bivalentes , Cobalto/química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Escherichia coli/genética , Hidrólisis , Serratia marcescens/genética , Espectrofotometría Ultravioleta , Espectroscopía de Absorción de Rayos X , Zinc/química , beta-Lactamasas/genéticaRESUMEN
Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K(i)=14µM) that is comparable to that of FBP (K(m)=2µM) or its inert analog TBP (K(i)=1µM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn(2+). The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn(2+) are not consistent with a strong interaction. To determine if Zn(2+)coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn(2+) coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn(2+) coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn(2+) coordination to binding of 8 to GlFBPA.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Giardia lamblia/enzimología , Animales , Sitios de Unión , Dihidroxiacetona Fosfato/química , Dihidroxiacetona Fosfato/metabolismo , Inhibidores Enzimáticos/química , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Cinética , Ligandos , Zinc/químicaRESUMEN
AQ-Pal14 is a 30-residue polypeptide that was designed to form an α-helical coiled coil that contains a metal-binding 4-pyridylalanine residue on its solvent-exposed surface. However, characterization of this peptide shows that it exists as a three-stranded coiled coil, not a two-stranded one as predicted from its design. Reaction with cobalt(III) protoporphyrin IX (Co-PPIX) produces a six-coordinate Co-PPIX(AQ-Pal14)(2) species that creates two coiled-coil oligomerization domains coordinated to opposite faces of the porphyrin ring. It is found that this species undergoes a buffer-dependent self-assembly process: nanometer-scale globular materials were formed when these components were reacted in unbuffered H(2)O, while millimeter-scale, rod-like materials were prepared when the reaction was performed in phosphate buffer (20 mM, pH 7). It is suggested that assembly of the globular material is dictated by the conformational properties of the coiled-coil forming AQ-Pal14 peptide, whereas that of the rod-like material involves interactions between Co-PPIX and phosphate ion.
Asunto(s)
Nanoestructuras/química , Péptidos/química , Péptidos/síntesis química , Protoporfirinas/química , Tampones (Química) , Cromatografía en Gel , Dicroismo Circular , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Modelos Moleculares , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría Infrarroja , Espectrometría Raman , Propiedades de Superficie , Ultracentrifugación , Espectroscopía de Absorción de Rayos XRESUMEN
Replacing the two Mn(2+) ions normally present in human Arginase I with Co(2+) resulted in a significantly lowered K(M) value without a concomitant reduction in k(cat). In addition, the pH dependence of the reaction was shifted from a pK(a) of 8.5 to a pK(a) of 7.5. The combination of these effects led to a 10-fold increase in overall catalytic activity (k(cat)/K(M)) at pH 7.4, close to the pH of human serum. Just as important for therapeutic applications, Co(2+) substitution lead to significantly increased serum stability of the enzyme. Our data can be explained by direct coordination of l-Arg to one of the Co(2+) ions during reaction, consistent with previously reported model studies. In vitro cytotoxicity experiments verified that the Co(2+)-substituted human Arg I displays an approximately 12- to 15-fold lower IC(50) value for the killing of human hepatocellular carcinoma and melanoma cell lines and thus constitutes a promising new candidate for the treatment of l-Arg auxotrophic tumors.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Arginasa/metabolismo , Arginasa/uso terapéutico , Cobalto/metabolismo , Manganeso/metabolismo , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Arginasa/química , Arginasa/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Espectroscopía de Absorción de Rayos XRESUMEN
We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-beta-lactamase (MbetaL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MbetaL move away from each other, by approximately 0.3 A after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 A. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 A, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with nitrocefin. This is the first direct structural information available on early stage dizinc metallo-beta-lactamase catalysis.
Asunto(s)
Biocatálisis , Movimiento (Física) , Zinc/química , Zinc/metabolismo , beta-Lactamasas/metabolismo , Absorción , Cristalografía por Rayos X , Análisis de Fourier , Análisis Espectral , Stenotrophomonas maltophilia/enzimologíaRESUMEN
In an effort to probe the structure, mechanism, and biochemical properties of metallo-beta-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, (1)H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.
Asunto(s)
Bacillus anthracis/enzimología , Cobalto/metabolismo , Zinc/metabolismo , beta-Lactamasas/metabolismo , Cinética , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Análisis Espectral , Especificidad por Sustrato , beta-Lactamasas/biosíntesisRESUMEN
ZnuA is the periplasmic Zn(2+)-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn(2+)-bound, and Co(2+)-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn(2+) with Co(2+) results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn(2+) periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn(2+) (estimated K (d) < 20 nM), Co(2+), Ni(2+), Cu(2+), Cu(+), and Cd(2+), but not Mn(2+). Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn(2+) substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metales/metabolismo , Periplasma/metabolismo , Absorción , Naftalenosulfonatos de Anilina/química , Sitios de Unión , Dicroismo Circular , Cobalto/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/citología , Fluorescencia , Modelos Moleculares , Estructura Secundaria de Proteína , Espectrofotometría Ultravioleta , Especificidad por Sustrato , Zinc/metabolismoRESUMEN
Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biological activity. The thermodynamic contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorimetry, potentiometry, and calorimetry to determine the thermodynamics of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation constants for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6 x 10(16), 1.5 x 10(15), or 2.5 x 10(13) M(-1), respectively. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, respectively, in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equivalent at physiological pH, DeltaG = -16.8 kcal/mol or a Ka = 2.0 x 10(12) M(-1). Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to determine the free energy required to fold the latter. Several proteins have identical Zn(II) affinities to GGG. That is, little, if any, of their Zn(II) binding energy is required to fold the protein, whereas some have affinities weakened by up to 5.7 kcal/mol; i.e., the Zn(II) binding energy is being used to fold the protein.
Asunto(s)
Péptidos/química , Dedos de Zinc , Secuencias de Aminoácidos , Calorimetría/métodos , Cisteína/química , Histidina/química , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Pliegue de Proteína , Espectrometría de Fluorescencia , Termodinámica , Zinc/químicaRESUMEN
Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.
Asunto(s)
Amidohidrolasas/metabolismo , Bacillus thuringiensis/enzimología , Percepción de Quorum , Cinética , Metales/metabolismo , Resonancia Magnética Nuclear BiomolecularRESUMEN
Substitution of natural nucleobases in PNA oligomers with ligands is a strategy for directing metal ion incorporation to specific locations within a PNA duplex. In this study, we have synthesized PNA oligomers that contain up to three adjacent bipyridine ligands and examined the interaction with Ni2+ and Cu2+ of these oligomers and of duplexes formed from them. Variable-temperature UV spectroscopy showed that duplexes containing one terminal pair of bipyridine ligands are more stable upon metal binding than their nonmodified counterparts. While binding of one metal ion to duplexes that contain two adjacent bipyridine pairs makes the duplexes more stable, additional metal ions lower the duplex stability, with electrostatic repulsions being, most likely, an important contributor to the destabilization. UV titrations showed that the presence of several bipyridine ligands in close proximity of each other in PNA oligomers exerts a chelate effect. A supramolecular chelate effect occurs when several bipyridines are brought next to each other by hybridization of PNA duplexes. EPR spectroscopy studies indicate that even when two Cu2+ ions coordinate to a PNA duplex in which two bipyridine pairs are next to each other, the two metal-ligand complexes that form in the duplex are far enough from each other that the dipolar coupling is very weak. EXAFS and XANES show that the Ni2+-bipyridine bond lengths are typical for [Ni(bipy)2]2+ and [Ni(bipy)3]2+ complexes.
Asunto(s)
2,2'-Dipiridil/química , Metales/metabolismo , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Emparejamiento Base , Sitios de Unión , Cobalto/química , Cobalto/metabolismo , Cobre/química , Cobre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , Metales/química , Níquel/química , Níquel/metabolismo , Volumetría , Rayos UltravioletaRESUMEN
The tetrahedral cobalt(II) complex [(Tp(Ph,Me))CoCl] (Tp(Ph,Me) = hydrotris(3,5-phenylmethylpyrazolyl)borate) was combined with several hydroxypyridinone, hydroxypyridinethione, pyrone, and thiopyrone ligands to form the corresponding [(Tp(Ph,Me))Co(L)] complexes. X-ray crystal structures of these complexes were obtained to determine the mode of binding for each ligand L. The structures show that the [(Tp(Ph,Me))Co(L)] complexes are pentacoordinate complexes, with a general tendency toward square pyramidal geometry. The electronic, EPR, and paramagnetic NMR spectroscopy of the [(Tp(Ph,Me))Co(L)] complexes have been examined. The frozen-solution EPR spectra are indicative of pentacoordination in frozen solution, while the NMR indicates some dynamics in ligand binding. The findings presented here suggest that [(Tp(Ph,Me))Co(L)] complexes can be used as spectroscopic references for investigating the mode of inhibitor binding in metalloproteinases of medicinal interest. Potential limitations when using cobalt(II) model complexes are also discussed.
Asunto(s)
Cobalto/química , Metaloproteinasas de la Matriz/química , Compuestos Organometálicos/síntesis química , Inhibidores de Proteasas/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón/métodos , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Sensibilidad y Especificidad , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of approximately 100 m(-1) cm(-1), which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M(S) = |+/-1/2), E/D = 0.14, g(real(x,y)) = 2.37, and g(real(z)) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at approximately 2.0 angstroms. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75-84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate D-Ala-D-Ala is also five-coordinate.