Molecular Cloning, Heterologous Expression, and Characterization of Human Glyoxalase II

Ridderström, Marianne; Saccucci, Franca; Hellman, Ulf; Bergman, Tomas; Principato, Giovanni; Mannervik, Bengt

doi:10.1074/jbc.271.1.319

Cited by 72 publications

(67 citation statements)

References 29 publications

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“…Examination of Available HAGH mRNA Sequences-The first reported human cDNA sequence of HAGH (X90999) had 8 coding exons (5). However, the NCBI Reference Sequence for HAGH mRNA (NM_005326) contains an additional 500-bp exon at the start of the sequence (termed exon A in this report).…”

Section: Resultsmentioning

confidence: 99%

“…It has broad substrate specificity for glutathione thiol esters, hydrolyzing a number of these species (besides S-D-lactoylglutathione) to their corresponding carboxylic acids and reduced glutathione. Among these substrates, 2-hydroxy thiol esters appear to be hydrolyzed with greatest efficiency (5).…”

mentioning

confidence: 99%

“…1 been reported to prevent MG accumulation and advanced glycation end product formation under hyperglycemic conditions (16). In vertebrates there is a single gene encoding glyoxalase II, HAGH (hydroxyacylglutathione hydrolase) (5,17,18).…”

mentioning

confidence: 99%

“…In this paper, we demonstrate that extended transcripts from the human HAGH gene encode both cytosolic and mitochondrial isoforms of glyoxalase II, and that the previous mRNA sequence is incomplete. The cytosolic form originates by internal priming of protein synthesis from the initiating AUG codon previously identified in exon 1 (5). The mitochondrial form arises from initiation of translation at an upstream AUG codon encoded on an upstream exon that can be fused in-frame to exon 1.…”

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confidence: 99%

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

Cordell¹,

Futers

Grant

et al. 2004

Journal of Biological Chemistry

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In yeast and higher plants, separate genes encode the cytosolic and mitochondrial forms of glyoxalase II. In contrast, although glyoxalase II activity has been detected both in the cytosol and mitochondria of mammals, only a single gene encoding glyoxalase II has been identified. Previously it was thought that this gene (the hydroxyacylglutathione hydrolase gene), comprised 8 exons that are transcribed into mRNA and that the resulting mRNA species encoded a single cytosolic form of glyoxalase II. Here we show that this gene gives rise to two distinct mRNA species transcribed from 9 and 10 exons, respectively. The 9-exon-derived transcript encodes two protein species: mitochondrially targeted glyoxylase II, which is initiated from an AUG codon in a previously uncharacterized part of the mRNA sequence, and cytosolic glyoxalase II, which is initiated by internal ribosome entry at a downstream AUG codon. The transcript deriving from 10 exons has an in-frame termination codon between the two initiating AUG codons and hence only encodes the cytosolic form of the protein. Confocal fluorescence microscopy indicates that the mitochondrially targeted form of glyoxalase II is directed to the mitochondrial matrix. Analysis of glyoxalase II mRNA sequences from a number of species indicates that dual initiation from alternative AUG codons is conserved throughout vertebrates.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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

Cordell¹,

Futers

Grant

et al. 2004

Journal of Biological Chemistry

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“…The GLX2-2 gene has been successfully cloned and overexpressed in Escherichia coli yielding protein concentrations as high as 100 M (18). Early biochemical studies suggested that glyoxalase II, unlike glyoxalase I, does not require bound metal ions for activity (13,15,17,19). However, through the analysis of recombinant GLX2-2, we demonstrated that glyoxalase II is a Zn(II)-requiring enzyme (18).…”

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confidence: 99%

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

Zang

Hollman

Crawford

et al. 2001

Journal of Biological Chemistry

123

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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...

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Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

et al. 2000

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Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria. Proteins 2000;41:33–39. © 2000 Wiley‐Liss, Inc.

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Molecular Cloning, Heterologous Expression, and Characterization of Human Glyoxalase II

Cited by 72 publications

References 29 publications

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

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

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

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

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