Role of the Zn\u3csub\u3e1\u3c/sub\u3e and Zn\u3csub\u3e2\u3c/sub\u3e Sites in Metallo-β-lactamase L1

In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme−product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate

Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions

Metallo-β-lactamases are responsible for conferring antibiotic resistance on certain pathogenic bacteria. In consequence, the search for inhibitors that may be useful in combating antibiotic resistance has fueled much study of the active sites of these enzymes. There exists circumstantial evidence that the binding of substrates and inhibitors to metallo-β-lactamases may involve binding to the organic part of the molecule, in addition to or prior to binding to one or more active site metal ions. It has also been postulated that a conformational change may accompany this putative binding. In the present study, electron paramagnetic resonance spectrokinetic study of a spin-labeled variant of the class B2 metallo-β-lactamase ImiS identified movement of a component residue on a conserved α-helix in a catalytically competent time upon formation of a transient reaction intermediate with the substrate imipenem. In a significant subpopulation of ImiS, this conformational change was not associated with substrate binding to the active site metal ion but, rather, represents a distinct step in the reaction with ImiS. This observation has implications regarding the determinants of substrate specificity in metallo-β-lactamases and the design of potentially clinically useful inhibitors

\u3cem\u3eArabidopsis thaliana\u3c/em\u3e GLX2-1 Contains a Dinuclear Metal Binding Site, but Is Not a Glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV–visible, EPR, and 1H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV–visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in 1H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg246 binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-β-lactamase superfamily

Spectroscopic Studies on \u3cem\u3eArabidopsis\u3c/em\u3e ETHE1, a Glyoxalase II-like Protein

ETHE1 (ethylmalonic encephalopathy protein 1) is a β-lactamase fold-containing protein that is essential for the survival of a range of organisms. In spite of the apparent importance of this enzyme, very little is known about its function or biochemical properties. In this study Arabidopsis ETHE1 was over-expressed and purified and shown to bind tightly to 1.2 ± 0.2 equivalents of iron. 1H NMR and EPR studies demonstrate that the predominant oxidation state of Fe in ETHE1 is Fe(II), and NMR studies confirm that two histidines are bound to Fe(II). EPR studies show that there is no antiferromagnetically coupled Fe(III)Fe(II) center in ETHE1. Gel filtration studies reveal that ETHE1 is a dimer in solution, which is consistent with previous crystallographic studies. Although very similar in terms of amino acid sequence to glyoxalase II, ETHE1 exhibits no thioester hydrolase activity, and activity screening assays reveal that ETHE1 exhibits low level esterase activity. Taken together, ETHE1 is a novel, mononuclear Fe(II)-containing member of the β-lactamase fold superfamily

Motion of the Zinc Ions in Catalysis by a Dizinc Metallo-β-Lactamase

We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-β-lactamase (MβL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MβL move away from each other, by ∼0.3 Å after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 Å. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 Å, 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-β-lactamase catalysis

Direct Evidence That the Reaction Intermediate of Metallo-β-lactamase L1 Is Metal Bound

In an effort to probe the structure of the reaction intermediate of metallo-β-lactamase L1 when reacted with nitrocefin and other β-lactams, time-dependent absorption and rapid-freeze-quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme. When using nitrocefin as the substrate, time-dependent absorption spectra demonstrate that Co(II)-substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. RFQ-EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)\u27s in the enzyme that is consistent with a change in the coordination sphere of this metal ion. This observation provides evidence that the reaction intermediate is a metal-bound species. RFQ-EPR studies also demonstrate that other β-lactams, such as cephalothin, meropenem, and penicillin G, proceed through an electronically similar complex and that the role of metal is similar in all cases. EPR spectroscopy has also identified distinct product-bound species of L1, indicating that reversible product binding must be considered in all future kinetic mechanisms. Consideration of the time-dependent optical and EPR studies in light of available crystallographic information indicates the intimate involvement of the metal ion in the Zn2-binding site of L1 in the hydrolytic reaction

Probing the Reaction Mechanism of the D-ala-D-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the D-ala-D-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (ε550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts

Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity

AbstractCytosolic glyoxalase II from Arabidopsis thaliana, GLX2-2, was overexpressed and purified to homogeneity using Q-sepharose chromatography. MALDI-TOF mass spectrometry studies indicated a molecular weight of 28 767 Da. Using steady-state kinetics studies, the purified enzyme exhibited a Km of 660±100 μM and a kcat of 484±92 s−1 at 37°C. Metal analyses demonstrated that the enzyme binds 2.1±0.5 moles of Zn(II) per monomer; the binding of Zn(II) is essential for enzyme viability and activity. Sequence comparison of glyoxalase II enzymes from human, A. thaliana, and yeast and the metallo-β-lactamases reveal that all metal binding ligands of the metallo-β-lactamases are conserved in glyoxalase II enzymes, suggesting that all glyoxalase II enzymes are Zn(II) metalloenzymes. These results and their implications are discussed in light of previous studies on glyoxalase II, and an active site for the glyoxalase II enzymes is proposed

Structural Studies on a Mitochondrial Glyoxalase II

Glyoxalase 2 is a β-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ± 0.15 eq of iron and 1.31 ± 0.05 eq of Zn(II) and to exhibit kcat and Km values of 129 ± 10 s-1 and 391 ± 48 μm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. 1H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II)

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 ∼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 = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(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 ∼ 2.0 Å. 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