Yeast Glyoxalase I Is a Monomeric Enzyme with Two Active Sites

Frickel, Eva-Maria; Jemth, Per; Widersten, Mikael; Mannervik, Bengt

doi:10.1074/jbc.m005760200

Cited by 51 publications

(55 citation statements)

References 29 publications

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“…The high degree of sequence similarity and the conservation of the predicted metal-binding residues between the trypanosomatid and the E. coli GLO1 enzymes suggest that the parasite enzymes all might be nickel-dependent. However, the cofactor requirement of a GLO1 enzyme cannot be predicted from sequence alone, because the zinc-dependent Pseudomonas putida and S. cerevisiae enzymes have identical metal-binding residues as those of the E. coli enzyme (31,32). Thus, the unambiguous demonstration of the requirement of the L. major GLO1 for nickel, but not zinc, by metal analysis and reconstitution is important in confirming the unique relationship of these eukaryotic glyoxalases to the E. coli enzyme.…”

Section: Discussionmentioning

confidence: 99%

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Vickers

Greig

Fairlamb

2004

Proc. Natl. Acad. Sci. U.S.A.

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Glyoxalase I forms part of the glyoxalase pathway that detoxifies reactive aldehydes such as methylglyoxal, using the spontaneously formed glutathione hemithioacetal as substrate. All known eukaryotic enzymes contain zinc as their metal cofactor, whereas the Escherichia coli glyoxalase I contains nickel. Database mining and sequence analysis identified putative glyoxalase I genes in the eukaryotic human parasites Leishmania major, Leishmania infantum, and Trypanosoma cruzi, with highest similarity to the cyanobacterial enzymes. Characterization of recombinant L. major glyoxalase I showed it to be unique among the eukaryotic enzymes in sharing the dependence of the E. coli enzyme on nickel. The parasite enzyme showed little activity with glutathione hemithioacetal substrates but was 200-fold more active with hemithioacetals formed from the unique trypanosomatid thiol trypanothione. L. major glyoxalase I also was insensitive to glutathione derivatives that are potent inhibitors of all other characterized glyoxalase I enzymes. This substrate specificity is distinct from that of the human enzyme and is reflected in the modification in the L. major sequence of a region of the human protein that interacts with the glycyl-carboxyl moiety of glutathione, a group that is conjugated to spermidine in trypanothione. This trypanothione-dependent glyoxalase I is therefore an attractive focus for additional biochemical and genetic investigation as a possible target for rational drug design.methylglyoxal ͉ D-lactate ͉ glutathione ͉ nickel

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Section: Discussionmentioning

confidence: 99%

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Vickers

Greig

Fairlamb

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Glass beads and cell debris were sedimented by centrifugation at 16,000 ϫ g for 5 min. The protein supernatant was removed and subsequently dialyzed overnight in 10 mM potassium phos- Enzyme activity assays for LGL were performed in a manner similar to the protocol of Frickel et al (7). Briefly, the reaction substrate, hemithioacetal, was prepared by incubating 4 mM each of reduced glutathione and methylglyoxal in 50 mM sodium phosphate buffer (pH 6.6) for 10 min at 37°C, and its concentration was determined spectrophotometrically (E 240 ϭ 0.44 mM Ϫ1 cm Ϫ1 ).…”

Section: Methodsmentioning

confidence: 99%

“…LGL is involved in methylglyoxal detoxification via the formation of S-D-lactoylglutathione from the hemimercaptal adduct that is formed nonenzymatically between glutathione and the 2-oxoaldehyde methylglyoxal (7). Glyoxalase II then converts S-D-lactoylglutathione into reduced glutathione and D-lactate (7). An examination of the S. mutans UA159 genome does not reveal the presence of a glyoxalase II homologue, suggesting an alternate pathway by which S-D-lactoylglutathione is neutralized.…”

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confidence: 99%

“…The formation of methylglyoxal occurs via enzymatic production during glycolysis from the fragmentation of triose phosphates (4,10). LGL is involved in methylglyoxal detoxification via the formation of S-D-lactoylglutathione from the hemimercaptal adduct that is formed nonenzymatically between glutathione and the 2-oxoaldehyde methylglyoxal (7). Glyoxalase II then converts S-D-lactoylglutathione into reduced glutathione and D-lactate (7).…”

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confidence: 99%

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Involvement of the Detoxifying Enzyme Lactoylglutathione Lyase in Streptococcus mutans Aciduricity

2007

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Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in S. mutans. Proteome analysis of S. mutans demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to S-D-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in S. mutans. An S. mutans isogenic mutant defective in lgl (LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of lgl resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for S. mutans UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of lgl conducted by quantitative real-time PCR revealed that lgl was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that lgl was induced during acidic growth (ϳ3.5-fold) and following acid adaptation (ϳ2-fold).This study demonstrates that in S. mutans, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.Dental caries is one of the most prevalent infectious dental diseases afflicting humans. The microorganism most strongly associated with human dental caries is Streptococcus mutans, a normal inhabitant of dental plaque. Two of the foremost cariogenic determinants of S. mutans are its acidogenicity and aciduricity (reviewed by Banas [3]). These properties are the ability to produce acid end products from the metabolism of dietary carbohydrates and the ability to survive and grow at low pH, respectively. Acidogenicity and aciduricity allow S. mutans to dramatically drop the pH within the dental plaque biofilm, which can promote the development and progression of carious lesions via its sustained dominance and acid production via glycolysis. The feast-or-famine conditions generated by the host diet can result in rapid increases in sugar that must quickly be removed from the environment. Likewise, the glycolytic intermediates must quickly be removed from the cell due to their toxicity.Lactoylglutathione lyase (LGL) is an enzyme involved in the detoxification of methylglyoxal, a highly toxic electrophilic glycolytic by-product that reacts with and inactivates intracellular macromolecules, including both proteins and nucleic acids (9, 10). Therefore, its rapid degradation is v...

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“…5). Yeast and P. falciparum GlxI, for example, are large single polypeptide enzymes, wherein the N terminus resembles its C-terminal domain, and form two active sites within one polypeptide chain (48,66). The N-terminal domain in these large GlxI likely resembles one subunit of the small GlxI, which consists of two domains (the N-and C-terminal domains).…”

Section: Discussionmentioning

confidence: 99%

Structural Variation in Bacterial Glyoxalase I Enzymes

Suttisansanee

Lau

Lagishetty

et al. 2011

Journal of Biological Chemistry

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The glyoxalase system catalyzes the conversion of toxic, metabolically produced ␣-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn 2؉ and non-Zn 2؉ activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Grampositive bacteria that has been fully characterized as to its threedimensional structure and its detailed metal specificity. It is a Ni 2؉ /Co 2؉ -activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn 2؉ -bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements. Methylglyoxal (MG),3 an ␣-ketoaldehyde produced from a number of different enzymes and pathways, including triosephosphate isomerase, amino acid degradation, and acetoneconverting monooxygenases, is found in both prokaryotic and eukaryotic organisms (1-4). MG is a cytotoxic compound that can be produced to a level as high as 0.4 mM per cell per day, leading to protein synthesis inhibition, adduct formation with proteins, DNA, and RNA and can promote advanced glycation end products (5-10).A major contributing pathway involved in the detoxification of MG is the glyoxalase system. This two-enzyme system consists of glyoxalase I (GlxI) and glyoxalase II (GlxII) that convert ␣-ketoaldehydes into their corresponding 2-hydroxycarboxylic acids (D-lactate in the case of methylglyoxal), using an intracellular thiol as a cofactor/cosubstrate (Fig. 1). The first enzyme, GlxI (S-D-lactoylglutathione methylglyoxal lyase (isomerizing), EC 4.4.1.5), converts a hemithioacetal, the product of the nonenzymatic reaction between MG and a thiol, such as glutathione (GSH), to S-D-lactoylglutathione. GlxI is a metalloenzyme that can be divided into two classes, Zn 2ϩ activation (i.e. Homo sapiens GlxI (11)) and non-Zn 2ϩ activation (being selectively Ni 2ϩ /Co 2ϩ -activated, i.e. Escherichia coli GlxI (12)). It is likely that almost all Gram-negative prokaryotes possess Ni 2ϩ /Co 2ϩ -activated GlxI with the...

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Yeast Glyoxalase I Is a Monomeric Enzyme with Two Active Sites

Cited by 51 publications

References 29 publications

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Involvement of the Detoxifying Enzyme Lactoylglutathione Lyase in Streptococcus mutans Aciduricity

Structural Variation in Bacterial Glyoxalase I Enzymes

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