Glyoxalase 2 is a ␤-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 1 H 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 k cat and K m values of 129 ؎ 10 s ؊1and 391 ؎ 48 M, 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)Zn(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.1 H 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).The glyoxalase system consists of two enzymes, lactoylglutathione lyase (glyoxalase I, GLX1) 2 and hydroxyacylglutathione hydrolase (glyoxalase II, GLX2), that act coordinately to convert a variety of ␣-ketoaldehydes into hydroxy acids in the presence of glutathione (1). Aromatic and aliphatic ␣-ketoaldehydes react spontaneously with glutathione to form thiohemiacetals, which are converted to S-(2-hydroxyacyl)glutathione derivatives by GLX1. GLX2 hydrolyzes S-(2-hydroxyacyl)glutathione derivatives to regenerate glutathione and produce hydroxy acids. Glyoxalase I, a Zn(II) or Ni(II) metalloprotein, can utilize a number of ␣-ketoaldehydes (2). However, the primary physiological substrate of the enzyme is thought to be methylglyoxal (MG), a cytotoxic and mutagenic compound that is formed primarily as a by-product of carbohydrate and lipid metabolism (3, 4). MG can react with DNA to form modified guanylate residues (5) and interstrand cross-links (6). It also reacts with proteins to form glycosylamine derivatives of arginine and lysine and hemithioacetals with cysteines (7).Cells with high glycolytic rates exhibit high rates of methylglyoxal formation and increased levels of GLX1 activity. For instance, increased levels of GLX1 and GLX2 RNA and protein have been detected in tumor cells, including breast carcinoma cells (8). Because selec...

In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and presteady state kinetic and rapid-freeze quench 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. Sitedirected 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 milliseconds that is assigned to a reaction intermediate that has a 5-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 milliseconds. Taken together, these results allow for a reaction mechanism to be offered for the B2 metallo-β-lactamases and demonstrates that the mononuclear Zn(II)-and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.Bacterial resistance to antibiotics is a growing clinical concern (1,2). Zn(II)-containing β-lactamases (metallo-β-lactamases, MβL's) contain 1-2 moles of Zn(II) per mole of enzyme, hydrolyze all known cephalosporins, carbapenems and penicillins, are not inhibited by clavulanic acid and other classical β-lactamase inhibitors, and have no known clinically-useful inhibitor towards them (3,4). Previous studies have shown that there is significant structural and mechanistic diversity among the MβL's, leading to the grouping of the enzymes into three distinct subclasses: B1, B2, and B3 (5,6). Sequence identity ranges from 25-40% between members in one subclass and from 10-20% between members in different subclasses. Subclass B1 enzymes have been found in strains of Bacillus, Bacteroides, Pseudomonas, Serratia, and Chryseobacterium, and subclass B3 enzymes have been found in strains of Stenotrophomonas, Legionella, Fluoribacter, Janthinobacterium and Caulobacter (3,4). Enzymes from the B1 and B3 subclasses have broad substrate profiles and require two Zn(II) † This work was supported by the National Institutes of Health (GM40052 to MWC; AI056231 to BB, and EB001980 to the Medical College of Wisconsin).*To whom correspondence should be addressed: M. W. Crowder, e-mail: crowdemw@muohio.edu, phone: (513) 529-7274, fax: (513) 529-5715. NIH Public Access Author ManuscriptBiochemistry. A...

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.