X-ray absorption spectroscopy of metal site speciation in the metallo-β-lactamase BcII from Bacillus cereus.

Cobalt and zinc binding by the subclass B1 metallo-β-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.

Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.

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.

Structural and kinetic studies on metallo-β-lactamase IMP-1.

In an effort to probe for metal binding to metallo-β-lactamase (MβL) 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.

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

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).

Nanometer to millimeter scale peptide-porphyrin materials.

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.

Replacing Mn(2+) with Co(2+) in human arginase i enhances cytotoxicity toward l-arginine auxotrophic cancer cell lines.

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.

Motion of the zinc ions in catalysis by a dizinc metallo-beta-lactamase.

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.

Differential binding of Co(II) and Zn(II) to metallo-beta-lactamase Bla2 from Bacillus anthracis.

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.

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli.

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.

Deducing the energetic cost of protein folding in zinc finger proteins using designed metallopeptides.

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.

Metal binding to bipyridine-modified PNA.

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.

The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect.

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.

Model complexes of cobalt-substituted matrix metalloproteinases: tools for inhibitor design.

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.

A five-coordinate metal center in Co(II)-substituted VanX.

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.