Structural Studies on a Mitochondrial Glyoxalase II

Abstract

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...

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

“…16,17 GLX2-5 from A. thaliana mitochondria was crystallized as a dimer held together by a number of electrostatic interactions and with the C-termini buried in the dimeric interface. 18 However, it did not prove possible to confirm dimerization in solution, and thus it was concluded that it must be a crystallization artifact. The overall structure of YcbL reveals the presence of the ab/ba fold that structurally defines the metallo-b-lactamase family of proteins, with the addition of an a-helix [residues 87-103, Fig.…”

Spectroscopic studies on Arabidopsis ETHE1, a glyoxalase II-like protein

“…GLX2 enzymes have been shown to bind iron, zinc, and manganese; therefore, ETHE1 was analyzed for all three of these metals and also copper [11,23]. The preparation of ETHE1 in rich medium containing no added metal ions resulted in an isolated enzyme that bound 0.2 equivalents of iron and no other metal ions at levels detectable with ICP-AES.…”

“…Crystal structure comparisons between Arabidopsis ETHE1 [10] and Arabidopsis GLX2-5, a mitochondrial GLX2 enzyme [11], revealed that while the proteins show only 13% sequence identity, ETHE1 is structurally very similar to GLX2 [12]. ETHE1 and GLX2 enzymes contain a very similar metal binding domain and the β-lactamase fold consisting of two central mixed β-sheets surrounded on both sides by helices [12,13].…”

“…The NMR data were surprising, since we initially predicted that ETHE1 would have a metal binding site similar to those in the GLX2 family. NMR spectra from GLX2-5, which can contain a dinuclear iron center, shows at least eight paramagnetically-shifted resonances in between 110 and −30 ppm that correspond to protons on ligands bound to a Fe(III)Fe(II) antiferromagnetically-coupled center [11]. The recent crystal structure of ETHE1 demonstrated that His74 (His56 in GLX2-5) may not be in a position to coordinate a bound metal ion [12], which is consistent with NMR spectrum of ETHE1.…”

Comparative Structure Modeling of Glyoxalase II Protein of Oryza sativa

“…As a first step in understanding the structure and function of mitochondrial gly II enzymes, a mitochondrial isozyme of gly II from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1 H NMR spectroscopies, and X-ray crystallography (Marasinghe et al, 2005). Naturally occurring homologous proteins have similar protein structure.…”