Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

Abstract

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or antiprotozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.The glyoxalase system consists of two enzymes that convert cytotoxic 2-oxoaldehydes into hydroxy acids utilizing the cofactor glutathione. Under physiological conditions, glyoxalase I (lactoylglutathione lyase) catalyzes the formation of S-D-lactoylglutathione (SLG) 1 in the presence of methylglyoxal and glutathione (1). Methylglyoxal, a cytotoxic compound that can inactivate proteins, modify guanylate residues in DNA, and create interstrand cross-links (2-4), is formed as a by-product of several biochemical reactions including that of triose-phosphate isomerase (2). Glyoxalase II (hydroxyacylglutathione hydrolase) hydrolyzes SLG to form D-lactic acid and regenerate glutathione (1). SLG is also known to exhibit cytotoxic effects through the inhibition of DNA synthesis (5). Therefore, the glyoxalase system is thought to play a major role in chemical detoxification (5, 6).The glyoxalase system has been the subject of intense study because of its potential implications in anti-protozoal and antitumor drug design (6 -8). Increased cellular levels of methylglyoxal and glyoxalase activity are associated with tumor cells, the malaria parasite, and several complications associated with diabetes (6). Inhibitors of glyoxalase I and glyoxalase II have been designed as potential anti-tumor agents; however, all inhibitors tested to date exhibited problems that limited their therapeutic usefulness (6 -9). It should be noted that these inhibition studies were all conducted with little or no information on the active site structure or the kinetic mechanism of the enzyme. Therefore, it is clear that a more detailed understanding of the active site of glyoxalase II and its reaction mechanism is essential to the rational design an...

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

“…This ability for catalysis to proceed with similar efficiency in the presence of different metal ions has also been observed in other bacterial GLX2 enzymes. 11,20 It may also indicate that the extensive hydrogen bonding network of liganding residues is able to undergo considerable flexibility in accommodating different metal ions, which is perhaps reflective of the requirement on the part of the organism to rapidly adapt to different environmental conditions.…”

“…Both histidine ligands form contacts through the Ne2 atom, in full agreement with the His-binding mode observed among the metallo-b-lactamase superfamily members. 11,17,20 One common structural feature of enzymes that have a metallo-b-lactamase fold is an extensive Hbonding network around the metal-binding ligands in the form of a so-called ''second shell'' of interactions. As YcbL represents the first example of a GLX2 enzyme with a single metal center, it is informative to compare the geometries of the secondshell amino acid metal ligands.…”

“…All family members also contain a binuclear metal binding site, with the zinc b-lactamases containing either a mono or dizinc center. [7][8][9][10] GLX2s contain a mixed zinc-iron binuclear center, 11 whereas the A-type flavoproteins contain a diiron site. 12 Structural alignment suggests that it is the subtle variation in the type of the ligand-coordinating amino acid that provides the metal specificity.…”

Glyoxalase II of African Trypanosomes Is Trypanothione-dependent

“…Glyoxalases II contain the highly conserved metal binding motif THXHXDH (23). In total, five His and two Asp residues interact directly with two metal ions as shown in the threedimensional structure of human glyoxalase II (24).…”

The Human Hydroxyacylglutathione Hydrolase (HAGH) Gene Encodes Both Cytosolic and Mitochondrial Forms of Glyoxalase II

“…Thus in A. thaliana five genes have been identified, three that appear to encode mitochondrial proteins (GLX2-1, GLX2-4, and GLX2-5), one that encodes a cytosolic protein (GLX2-2), and one whose function is currently uncharacterized (GLX2-3) (10,13). An apparent homologue of one of the mitochondrial forms (AY054407 from Oryza sativa) has been overexpressed in tobacco and increases resistance to salinity (14).…”