David L. Tierney

Replacing Mn2+ with Co2+ 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 7.5. 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.

Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA.

The Metallo-β-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs.

Differential Binding of Co(II) and Zn(II) to Metallo-β-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. 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.

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM‑1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data.

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. 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.

Mechanistic Studies of the O2-Dependent Aliphatic Carbon−Carbon Bond Cleavage Reaction of a Nickel Enolate Complex

The mononuclear nickel(II) enolate complex [(6-Ph(2)TPA)Ni(PhC(O)C(OH)C(O)Ph]ClO(4) (I) was the first reactive model complex for the enzyme/substrate (ES) adduct in nickel(II)-containing acireductone dioxygenases (ARDs) to be reported. In this contribution, the mechanism of its O(2)-dependent aliphatic carbon-carbon bond cleavage reactivity was further investigated. Stopped-flow kinetic studies revealed that the reaction of I with O(2) is second-order overall and is ∼80 times slower at 25 °C than the reaction involving the enolate salt [Me(4)N][PhC(O)C(OH)C(O)Ph]. Computational studies of the reaction of the anion [PhC(O)C(OH)C(O)Ph](-) with O(2) support a hydroperoxide mechanism wherein the first step is a redox process that results in the formation of 1,3-diphenylpropanetrione and HOO(-). Independent experiments indicate that the reaction between 1,3-diphenylpropanetrione and HOO(-) results in oxidative aliphatic carbon-carbon bond cleavage and the formation of benzoic acid, benzoate, and CO:CO(2) (∼12:1). Experiments in the presence of a nickel(II) complex gave a similar product distribution, albeit benzil [PhC(O)C(O)Ph] is also formed, and the CO:CO(2) ratio is ∼1.5:1. The results for the nickel(II)-containing reaction match those found for the reaction of I with O(2) and provide support for a trione/HOO(-) pathway for aliphatic carbon-carbon bond cleavage. Overall, I is a reasonable structural model for the ES adduct formed in the active site of Ni(II)ARD. However, the presence of phenyl appendages at both C(1) and C(3) in the [PhC(O)C(OH)C(O)Ph](-) anion results in a reaction pathway for O(2)-dependent aliphatic carbon-carbon bond cleavage (via a trione intermediate) that differs from that accessible to C(1)-H acireductone species. This study, as the first detailed investigation of the O(2) reactivity of a nickel(II) enolate complex of relevance to Ni(II)ARD, provides insight toward understanding the chemical factors involved in the O(2) reactivity of metal acireductone species.

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

Integrated paramagnetic resonance, utilizing electron paramagnetic resonance (EPR), NMR, and electron-nuclear double resonance (ENDOR), of a series of cobalt bis-trispyrazolylborates, Co(Tp ( x )) 2, are reported. Systematic substitutions at the ring carbons and on the apical boron provide a unique opportunity to separate through-bond and through-space contributions to the NMR hyperfine shifts for the parent, unsubstituted Tp complex. A simple relationship between the chemical shift difference (delta H - delta Me) and the contact shift of the proton in that position is developed. This approach allows independent extraction of the isotropic hyperfine coupling, A iso, for each proton in the molecule. The Co..H contact coupling energies derived from the NMR, together with the known metrics of the compounds, were used to predict the ENDOR couplings at g perpendicular. Proton ENDOR data is presented that shows good agreement with the NMR-derived model. ENDOR signals from all other magnetic nuclei in the complex ( (14)N, coordinating and noncoordinating, (11)B and (13)C) are also reported.

Stress Response and Virulence Functions of the Acinetobacter baumannii NfuA Fe-S Scaffold Protein

To successfully establish an infection, Acinetobacter baumannii must overcome the iron starvation and oxidative stress imposed by the human host. Although previous studies have shown that ATCC 19606(T) cells acquire iron via the acinetobactin-mediated siderophore system, little is known about intracellular iron metabolism and its relation to oxidative stress in this pathogen. Screening of an insertion library resulted in the isolation of the ATCC 19606(T) derivative 1644, which was unable to grow in iron-chelated media. Rescue cloning and DNA sequencing showed that the insertion inactivated a gene coding for an NfuA Fe-S cluster protein ortholog, without any effect on the expression of the acinetobactin system. The nfuA mutant was also more sensitive to hydrogen peroxide and cumene hydroperoxide than the parental strain. The iron chelation- and oxidative-stress-deficient responses of this mutant were corrected when complemented with either the ATCC 19606(T) parental allele or the Escherichia coli MG1655 nfuA ortholog. Furthermore, electron paramagnetic resonance (EPR) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analyses showed that the ATCC 19606(T) NfuA ortholog has iron-binding properties compatible with the formation of [Fe-S] cluster protein. Ex vivo and in vivo assays using human epithelial cells and Galleria mellonella, respectively, showed that NfuA is critical for bacterial growth independent of their capacity to acquire iron or the presence of excess of free iron. Taken together, these observations indicate that the A. baumannii NfuA ortholog plays a role in intracellular iron utilization and protection from oxidative-stress responses that this pathogen could encounter during the infection of the human host.

Biochemical, mechanistic, and spectroscopic characterization of metallo-β-lactamase VIM-2.

This study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.