Biochemical Journal

Review article

The glyoxalase pathway: the first hundred years… and beyond

Marta Sousa Silva, Ricardo A. Gomes, Antonio E. N. Ferreira, Ana Ponces Freire, Carlos Cordeiro


The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time. Its association to detoxification and anti-glycation defence are its presently accepted roles, since methylglyoxal exerts irreversible effects on protein structure and function, associated with misfolding. This functional defence role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic target, since its inhibition might lead to an increased methylglyoxal concentration and cellular damage. However, metabolic pathway analysis showed that glyoxalase effects on methylglyoxal concentration are likely to be negligible and several organisms, from mammals to yeast and protozoan parasites, show no phenotype in the absence of one or both glyoxalase enzymes. The aim of the present review is to show the evolution of thought regarding the glyoxalase pathway since its discovery 100 years ago, the current knowledge on the glyoxalase enzymes and their recognized role in the control of glycation processes.

  • glycation
  • glyoxalase
  • Maillard reaction
  • methylglyoxal


The observation that yeast cell-free extracts could catalyse the formation of ethanol and carbon dioxide from glucose heralded modern biochemistry. At the time, little was known of the chemical composition of living cells and much less so regarding the chemical reactions that enable life. Attempts to reproduce biochemical processes revealed that a few molecules might be intermediates in the process of fermentation. One such molecule was methylglyoxal, readily formed under alkaline conditions from glyceraldehyde or dihydroxyacetone. The discovery in 1913 of an enzymatic activity that converted methylglyoxal into lactic acid appeared to show the path from glucose to pyruvic acid or ethanol [1,2]. Initially termed ketonaldehydemutase, it was later that the designation of GLO (glyoxalase) became accepted. Methylglyoxal and GLO became a focal point in glycolysis. At that time, the accepted model for glycolysis consisted of a splitting of glucose into two trioses that form methylglyoxal which was then converted into lactate through the action of GLO [3]. Although this scheme held its own for a long time, several observations dismissed a glycolytic role for methylglyoxal and GLO. Indeed, it was soon recognized that GLO required glutathione as a co-factor, whereas glutathione has no effect on glycolysis [4,5].

The search for an enzymatic origin of methylglyoxal was hampered by the discovery that DHAP (dihydroxyacetone phosphate) and GAP (D-glyceraldehyde 3-phosphate) are chemically unstable in physiological conditions and produce methylglyoxal non-enzymatically [6]. Methylglyoxal was therefore assumed not to be a key metabolite, entering the realm of chemical artefacts.

In 1948 a puzzling observation was made by Hopkins and Morgan [7] of an isolated factor that increased the rate of the GLO-catalysed reaction. This observation was soon rationalized as indicating the presence of a second enzyme by Crook and Law in 1950 [8], although the proof that two enzymes were required to produce lactate from methylglyoxal came later from Racker [9] with subsequent confirmation by Crook and Law [10]. Racker [9] showed that GLO activity could be accounted for by the action of two enzymes that he partially purified and named GLO1 and GLO2. He also showed the existence of an intermediate with specific spectral characteristics and demonstrated that both GLOs catalysed virtually irreversible reactions (Figure 1). This was one of the most relevant works in the field, being one of the most fruitful demonstrations of the power of spectrophotometric methods in enzymology. It was also the basis of the first specific enzymatic assay for glutathione. Later, the lactic acid formed through the action of the GLO pathway was found to be D-lactate, ruling out methylglyoxal and GLOs from glycolysis [11]. This raised the question of the physiological role of the GLO pathway since this two-enzyme system had no physiological substrate and led to a dead-end product.

Figure 1 The GLO system

This enzymatic pathway comprises two enzymes (GLO1 and GLO2) responsible for the glutathione-dependent catabolism of methylglyoxal, producing D-lactate.

In the late sixties, Szent-Gyorgyi [12,13] proposed an electronic theory of cancer on the basis of the regulation of the conducting properties of proteins as evidenced by quantum biochemistry studies. A reversible reaction with dicarbonyls would lead to a change in protein conductivity. Since glutathione and GLO activity could be related to cell proliferation, a retine–promine theory was proposed whereby retine, a dicarbonyl, would inhibit cell division, and promine, GLO and glutathione would enhance cell division [13]. Although this hypothesis didn't hold true it was the first time that the GLO pathway and protein modifications by dicarbonyls had a proposed regulatory role. Later, on the basis of the observation that methylglyoxal and other dicarbonyls are highly reactive and that glutathione-dependent enzymes serve detoxification roles, Mannervik [14] linked the GLO enzymes to the elimination of toxic dicarbonyls. This is the most currently accepted role of the GLO enzymes, particularly in the context of glycation. Glycation, the irreversible modification of amino groups in biological molecules, mainly proteins, nucleic acids and membrane lipids, is discussed below. Remarkably, the reaction of carbonyls with amino and guanidino groups was discovered long ago [1517] and has been linked to food processing long before its relevance was considered in biochemistry and medicine. Nevertheless, this is a crucial area of research in the context of human diseases, mainly diabetes and neurodegenerative conformational disorders, such as ATTR (transthyretin amyloidosis), Parkinson's disease and Alzheimer's disease.


Methylglyoxal is a ubiquitous product of cellular metabolism being therefore present in all cells, either under normal or pathological conditions. Enzymatic and non-enzymatic routes are known to produce methylglyoxal. The rate of methylglyoxal formation depends on the organism, tissue, cell metabolism and physiological conditions.

Non-enzymatic sources of methylglyoxal

Research in methylglyoxal biochemistry, GLOs and glycation has been hampered until recently by limitations in analytical methods. Specific and sensitive methods for the assay of methylglyoxal are crucial to investigate its formation pathways and catabolism. The most widely accepted ones are based on its reaction with aromatic o-diamines, such as 1,2-diaminobenzene or 1,2-diamino-4,5-dimethoxybenzene. Coupled to HPLC or LC-MS (liquid chromatography MS) analysis, these methods have proven effective, sensitive and specific [1820]. Great care in the derivatization reactions must be exerted to prevent non-enzymatic conversion of triose phosphates into methylglyoxal and it is probable that, in some cases, methylglyoxal concentration has been grossly overestimated [21].

Undoubtedly, the major pathway for methylglyoxal formation in eukaryotic cells arises from the glycolytic bypass by the non-enzymatic decomposition of the triose phosphates DHAP and GAP [2224]. Incidentally, this reaction has been known since the mid-1930s [6], but was considered an artefact of no physiological significance. The reaction was investigated in detail some years later, confirming the formation of methylglyoxal from the triose phosphates [25]. At a physiological pH, these triose phosphates have a high reactivity towards the loss of α-carbonyl protons, producing an enediolate phosphate, which has a low energy barrier for the phosphate group elimination [22]. Thus the substrate deprotonation to an enediolate phosphate intermediate followed by the spontaneous non-enzymatic β-elimination of the phosphate group leads to the formation of methylglyoxal (Figure 2) [25]. An estimate for the non-enzymatic methylglyoxal formation rate is 0.1 mM per day in rat tissues [25]. In the reaction catalysed by TIM (triose phosphate isomerase/GAP ketol-isomerase, EC., the stabilization of the enzyme-bound enediolate phosphate intermediate is therefore an absolute requirement to avoid degradation of the transition state intermediate into methylglyoxal. In fact, the enzyme-bound enediolate phosphate intermediate is protonated approximately 106-fold faster than it expels the phosphate group [24]. This is achieved by the interaction between the phosphate group of the enzyme-bound intermediate and a flexible loop of the enzyme [26,27]. The deletion of four amino acid residues within this TIM loop results in better catalysis of the elimination reaction, which generates methylglyoxal, than the normal isomerization reaction [28]. Unfortunately, the reaction with TIM is not perfect and the enediolate intermediate may leak from the active centre producing methylglyoxal in a paracatalytic reaction [24] (Figure 2). Nevertheless, the main source of methylglyoxal in biological systems is the non-enzymatic decomposition of triose phosphates [23].

Figure 2 Methylglyoxal formation from the triose phosphates DHAP and GAP

Triose phosphates are unstable molecules and the β-elimination reaction of the phosphate group from the common enediolate phosphate intermediate irreversibly yields methylglyoxal. The stabilization of this intermediate by TIM is essential to avoid methylglyoxal formation. However, enediolate phosphate intermediate can leak from the enzyme active site forming methylglyoxal in a paracatalytic reaction.

Other methylglyoxal formation routes include the oxidation of aminoacetone in the catabolism of L-threonine, mediated by the enzyme SSAO (semicarbazide-sensitive amine oxidase, EC [29], the oxidation of ketone bodies by myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC [30] and the oxidation of acetone by cytochrome P450 [substrate, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating or -epoxidizing), EC] [31,32]. In pathological conditions like ketosis and diabetic ketoacidosis, the oxidation of ketone bodies is likely to be an important source of methylglyoxal [33]. Other non-enzymatic sources of methylglyoxal are the Maillard [34] and lipoperoxidation [35] reactions.

Methylglyoxal synthase

The only known enzyme that specifically catalyses methylglyoxal formation is methylglyoxal synthase. This enzyme was found, until recently, only in bacteria [36,37] and catalyses the formation of methylglyoxal from the triose phosphate DHAP [37,38]. D-Lactate produced by the GLO system may then be converted into pyruvate by D-lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC, providing a glycolysis bypass for pyruvate formation from DHAP [36] (Figure 3). Interestingly, methylglyoxal synthase is co-operatively inhibited by inorganic phosphate and it was suggested that it plays a role in the regulation of glycolysis depending on the availability of intracellular inorganic phosphate [39]. In Desulfovibrio gigas, this bypass can account for 40% of the glycolytic flux [40]. Although the isolation of methylglyoxal synthase from the goat liver has been reported [41], the presence of this enzyme in eukaryotic cells has not yet been detected [23,42,43].

Figure 3 Methylglyoxal bypass of glycolysis

In micro-organisms, methylglyoxal synthase (MGS) catalyses the formation of methylglyoxal from the triose phosphate DHAP. D-Lactate produced by the glyoxalase system (GLO1 and GLO2) or GLO3 may then be converted into pyruvate by D-lactate dehydrogenase (DLDH), providing a bypass for pyruvate formation from DHAP via glycolysis. This enzymatic bypass depends on the availability of intracellular inorganic phosphate. The grey arrow represents the non-enzymatic formation of methylglyoxal.

Other α-oxoaldehydes

Besides methylglyoxal, other α-oxoaldehydes, substrates of the GLOs, have been found in living systems. Glyoxal, phenylglyoxal and hydroxy-pyruvaldehyde were the first, discovered in 1952 by Racker [44]. Glyoxal, another oxoaldehyde substrate for GLO1, originates from glucose degradation [34]. Remarkably, some methylglyoxal-related α-oxoaldehydes are glycolysis by-products, such as hydroxypyruvaldehyde and 3-phospho-hydroxy-methylglyoxal. The first one is formed through the enediol oxidation from dihydroxyacetone and glyceraldehyde [45,46]. Hydroxypyruvaldehyde is an oxidation product of glyceraldehyde, dihydroxyacetone or methylglyoxal [47], whereas its phosphorylated form originates from the reaction of DHAP with hydrogen peroxide [48]. Kethoxal (3-ethoxy-2-oxobutyraldehyde), a cytotoxic agent [49], is also an active substrate for GLO1 [50].


Methylglyoxal and the GLO pathway remain one of the most elusive challenges of modern biochemistry. Methylglyoxal is presently believed to be a toxic compound and the GLO pathway (Figure 1) would thus serve a detoxification role. GLO1 (lactoylglutathione methylglyoxallyase; EC and GLO2 (hydroxyacylglutathione hydrolase, EC comprise this thiol-dependent GLO system, responsible for the formation of D-lactate from the hemithioacetal formed non-enzymatically from methylglyoxal and glutathione [51]. The discovery in 1995 of a GLO3 in Escherichia coli [52] challenged the concept of the GLO system as a pathway.


GLO1 has been characterized in many different organisms, from mammals [53,54] to plants [55,56], yeast [53,5759], bacteria [6062] and protozoan parasites [6368]. Considered ubiquitous in all living cells, this enzyme is, however, absent in some organisms. For example, the protozoans Entamoeba histolytica, Giardia lamblia and Trypanosoma brucei do not have the GLO1 gene [69].

The reaction catalysed by GLO1 is virtually irreversible with the formation of the thioester S-D-lactoylglutathione from the methylglyoxal–glutathione hemithioacetal. Other glutathione-derived hemithioacetals formed with a different α-oxoaldehyde (like methylglyoxal, hydroxypyruvaldehyde, glyoxal and phenylglyoxal) are also substrates for GLO1 [70,71]. In almost all kinetic studies involving this enzyme, the substrate is hemithioacetal, previously formed in a non-enzymatic step between methylglyoxal and glutathione [59,65,7274]. However, since the three species are simultaneously in equilibrium, the kinetic studies involving this enzyme are quite difficult and the reaction mechanism for GLO1 is not straightforward [70,7577]. The single-substrate mechanism using the methylglyoxal–glutathione hemithioacetal was proposed in 1961 and was almost universally accepted without question [78]. Later, a mechanism of ordered reaction was discovered, with glutathione followed by the α-oxoaldehyde binding to the active site [79]. By performing a steady-state kinetic analysis of GLO1 from erythrocytes and yeast, the same authors proposed a random mechanism comprising the two hypothesis: GLO1 can react with one substrate, the hemithioacetal, or it can display a two-substrate ordered mechanism, with glutathione as the first substrate and methylglyoxal as the second [75,76]. These seminal papers were the first ones to report a goodness of fit mechanism to discriminate between different steady-state enzyme kinetic models. According to the authors, the intracellular enzyme may already be complexed with the thiol and reacts with methylglyoxal on the catalytic surface of the enzyme. These studies were seldom taken into account when studying GLO1 kinetics, the single-substrate mechanism being taken for granted [63,67,74,80,81]. To address this long-standing mystery, a novel approach for model discrimination in enzyme kinetics was used [82]. By optimizing the initial reaction conditions for enzyme and substrate concentrations that maximize the differences between models (quantified by the Kullback–Leibler distance), the best possible discrimination conditions were achieved. For GLO1 from yeast, the single-substrate model is not valid, the two substrate mechanism being the most probable one [82].

Concerning thiol specificity, GLO1 from trypanosomatids are an exception, using a bis(glutathione)–spermidine conjugate (trypanothione) as a substrate instead of glutathione (reviewed in [69]). In these parasites, trypanothione is the preferred substrate for the GLO pathway, although GLO1 can also catalyse the isomerization of the glutathione-derived hemithioacetal [64,67].

Structurally, GLO1 enzymes are homodimers containing a zinc or nickel metal centre, essential for catalysis, and a thiol-binding pocket located at the interface of the two subunits [54,60,61,64,68]. Notable exceptions include the monomeric enzymes with two functional active sites from Saccharomyces cerevisiae [57] and Plasmodium falciparum [66]. Interestingly, the two active sites from P. falciparum GLO1 are allosterically coupled and have different affinities for the glutathione-derived hemithioacetal, responding differently to lower or higher methylglyoxal intracellular concentrations [74,83]. Several structures of GLO1 proteins were determined, namely human (PDB codes 1QIN, 1QIP, 1BH5 and 1FRO [54,84,85]), E. coli (PDB codes 1F9Z, 1FA5, 1FA6, 1FA7 and 1FA8 [60]), Leishmania major (PDB code 2C21 [64]) and Leishmania infantum [68]. Two human structures in complex with transition state analogues (PDB codes 1QIN and 1QIP) allowed the identification of the glutathione-binding site and highlighted the importance of the metal in co-ordinating the enediolate intermediate [85]. As mentioned, the metal in GLO1 can be either zinc or nickel, depending on the organism (Figure 4). It is commonly accepted that eukaryotic GLO1 enzymes contain zinc, including human [54], yeast [53] and L. infantum ([68] and reviewed in [69]), whereas the prokaryotic enzymes have nickel, such as from the bacteria E. coli, Pseudomonas aeruginosa, Yersinia pestis and Neisseria meningitides [61]. Once again, there are some enzymes that escape this rule, like the zinc-dependent GLO1 from the prokaryotic Pseudomonas putida [86] and the nickel-containing GLO1s from the eukaryotes Trypanosoma cruzi [65] and L. major [64,80].

Figure 4 X-ray crystallographic structures of GLO1

Human (PDB code 1QIP) (A), L. major (PDB code 2C21) (B) and E. coli (PDB code 1F9Z) (C) GLO1. Metal-binding sites and secondary structure elements are shown. These proteins are homodimeric and the subunits are coloured gold and blue. Zinc atoms are coloured grey (A) and nickel atoms are blue (B and C). Metal ions are co-ordinated by residues from both subunits.

GLO1 seems to be regulated through phosphorylation of a threonine residue and a nitric oxide modification of a cysteine residue, but the real biological function of these post-translational modifications is still unknown [87].


GLO2 was characterized in several eukaryotes and prokaryotes, including humans [88,89], plants [90,91], yeast [58,59,92], bacteria [93,94] and protozoan parasites [67,9598]. Like GLO1, this enzyme is nearly ubiquitous in all living organisms, although there are reports of its absence in some mammals [99,100]. Interestingly, some protozoan parasites lacking GLO1 have two GLO2-encoding genes and in other organisms, such as S. cerevisiae and P. falciparum, there are two GLO2 enzymes (reviewed in [69]). Both GLO2 enzymes are active in P. falciparum parasites and have different cellular locations: one is cytosolic (cGLO2) and the other one is targeted to the apicoplast (tGLO2) [63]. In S. cerevisiae, there is one cytosolic enzyme, GLO2, and the other is mitochondrial (GLO4), an intriguing location since GLO1 is absent from yeast mitochondria [101]. The presence of this GLO4 and the detection of S-D-lactoylglutathione inside the mitochondria suggested that this could be an alternative pathway to obtain glutathione in this organelle [102].

GLO2 is highly specific for the glutathione moiety of the substrate, although it can react with other thioesters [51,103,104]. A mutation in the glutathione-binding domain at the C-terminus of human GLO2 resulted in a complete loss of activity [105]. GLO2 from trypanosomatids, like GLO1, is an exception regarding thiol-dependence. The enzyme hydrolyses S-D-lactoyltrypanothione and shows absolute specificity towards this substrate [67,95,96]. The trypanothione-derived thioester binds to the active site through the spermidine moiety and the substrate-binding pocket cannot accommodate the S-D-lactoylglutathione [96] (Figure 5). Replacing two residues in the parasite enzyme by the correspondent ones from the human homologue produced a mutant able to hydrolyse both thioesters [106]. These studies were possible due to the availability of protein structures from different organisms, including human (PDB codes 1QH3 and 1QH5 [88]), Arabidopsis thaliana (PDB codes 2Q42 and 1XM8 [107,108]), Salmonella typhimurium (PDB code 2QED [109]) and L. infantum (PDB codes 2P18 and 2P1E [96]). GLO2 are monomeric enzymes that contain a binuclear metal centre (with zinc or iron) co-ordinated by the highly conserved motif THxHxDH, common to all known GLO2s and to the zinc-dependent metallo-β-lactamases [88,91,96]. These metal ions are essential for substrate binding and for catalysis [91].

Figure 5 Glutathione and trypanothione specificity of GLO2

The metal co-ordination site and the molecular surface silhouette around the substrate-binding site are shown for the human (PDB code 1QH5) (A) and L. infantum (PDB code 2P18) (B) enzymes. The ligands glutathione (GSH) and spermidine (SPD), a moiety of the trypanothione molecule, are also shown. The presence of a binding pocket for the spermidine moiety in the trypanossomatid enzyme, absent in the human homologue, accounts for its trypanothione specificity.


GLO3 (EC catalyses the conversion of methylglyoxal into D-lactate without requiring glutathione or any other co-factor in E. coli [52]. This is an irreversible reaction with neither formation nor catabolism of S-D-lactoylglutathione (Figure 3). Although the assay method using 2,4-dinitrophenylhydrazine is not specific for methylglyoxal [51], no other carbonyls were present in the reaction performed with the purified enzyme. Additionally, the formation of D-lactate was confirmed using the specific enzymatic assay adapted from a D-lactate dehydrogenase enzymatic assay. However, this is not a very efficient enzyme with a kcat/Km value of 1.8×103 M−1·s−1 [110], when compared with GLO1, which has a kcat/Km value of 1.2×107 M−1·s−1 [111]. GLO3 increases its expression during the bacterial stationary growth phase and is regulated by rpoS (RNA polymerase sigma factor) [112]. This enzyme exhibits a higher activity than GLO1 and GLO2 and represents the main system for methylglyoxal detoxification in these cells [112,113]. Curiously, its expression is not induced in the presence of methylglyoxal in growth medium [113] or in mutants lacking GLO1 [62].

Recently, another function for this enzyme, as a heat-inducible molecular chaperone [named Hsp (heat-shock protein) 31], was reported [110]. E. coli Hsp31 (encoded by hchA) is a homodimer that interacts with early unfolding intermediates preventing aggregation [114]. The analysis of a crystal structure for this enzyme (there are currently five known structures: PDB codes 1N57, 1ONS, 1PV2, 1IZY and 1IZZ) revealed three potential active sites [115]: a hydrophobic substrate-binding site responsible for the chaperone activity; a potential protease-like catalytic site formed by Cys184, His185 and Asp213; and a 2-histidine-1-carboxylate motif, formed by His85, Glu90 and His122 co-ordinating a zinc ion, possibly related to another function for this protein [115]. This motif shares similarities with the metal-binding site from GLO1, with one histidine and one glutamic acid residue from each monomer co-ordinating the metal ion [54,72], and GLO2, with three histidine residues and one aspartic acid residue (THxHxDH; [88,91,96,116]) that could be the binding site for methylglyoxal.

Previously, GLO3 was only known in bacteria; however, recent studies have described the activity of a novel type of GLO able to convert methylglyoxal into lactate without the requirement for glutathione in human cells [117]. This enzyme is DJ-1 [also known as PARK7 (Parkinson disease protein 7)], a molecular chaperone associated with the early onset of Parkinson's disease. GLO3 activity was also found in its homologues from the mouse and Caenorhabditis elegans [117]. In comparison with human GLO1, which exhibits a kcat/Km value between 0.9×107 and 2.6×107 M−1·s−1 [73], DJ-1 has a lower efficiency in methylglyoxal catabolism (kcat/Km=2.0×103 M−1·s−1 [117]). It was proposed that DJ-1/GLO3 is a scavenger for reactive carbonyl species [117], but its relevance for methylglyoxal catabolism is still unknown. This 23 kDa protein is a dimer with a putative active site close to the dimer interface, in which the residues Cys106, His126 and Glu18 may play an important role in catalysis [118]. This catalytic triad, cysteine, histidine and glutamic acid/aspartic acid, is identical with the one at the putative protease catalytic site in E. coli Hsp31, also known as GLO3 [115], and the structures of both proteins are rather similar, sharing an evolutionarily conserved domain [119]. Curiously, the 25 kDa yeast protein YDR533Cp (HSP31/YDR533C) is a member of the DJ-1/ThiJ/PfpI superfamily and its closest relative is Hsp31 [120]. It is also dimeric, contains a putative metal-binding site and the conserved cysteine, histidine and gluatamic acid catalytic triad [120], but its physiological functions remain unknown.


Given its specificity for methylglyoxal and other oxo-aldehydes, the role of the GLO pathway is related to the biological importance of these compounds. The fact that they are among the strongest electrophiles found in biological systems suggests that this chemical reactivity must be taken into consideration to define the biological function of the GLO enzymes.

Protein glycation and AGEs (advanced glycation end-products)

Methylglyoxal is the most relevant and reactive glycation agent in vivo. Glycation is a complex series of parallel and sequential reactions, collectively called the Maillard reaction, whereby reactive aldehydes modify amino groups. Investigation of the glycation of amino groups, pioneered by the work of Louis Camille Maillard who studied the formation of ‘brown products’ resulting from the reaction between glucose and amino acids [121], led to studies of protein glycation by glucose in physiological systems. Protein glycation by glucose occurs by reaction with the N-terminal lysine amino acid side chain and forms the Amadori product Nϵ-fructosyl-lysine and N-terminal amino acid residue-derived fructosamines [122,123]. In 1968, the glycation of haemoglobin with glucose in vivo was described with the formation of the N-terminal fructosamine adduct [124]. The occurrence of this ‘glycated’ form of haemoglobin turned out to be of great clinical significance, since its relative concentration can be used to assess glycaemic control in diabetes patients [125]. The Amadori product undergoes a complex series of chemical reactions to yield stable irreversible bound adduct, the AGEs [126129]. CML [N-(carboxymethyl)lysine] and pentosidine were the first stable AGEs discovered [126,127]. Reactive dicarbonyl compounds, such as methylglyoxal, glyoxal and 3-deoxyglucosone, are also produced from glucose-induced glycation reactions [34]. In contrast with glucose, methylglyoxal and glyoxal are much more reactive and glycation is directed mainly to the guanidine groups of arginine residues. Given the high relevance of methylglyoxal-derived protein modifications, the term MAGE (methylglyoxal-derived AGEs) has been coined (Figure 6) [130]. Methylglyoxal reacts with arginine residues to form a hydroimidazolone derivate (MG-H, with three related structural isomers), argpyrimidine and THP (tetrahydropyrimidine). The reaction between lysine residues and methylglyoxal leads to the formation of CEL [Nϵ-(carboxyethyl)lysine] and MOLDs (methylglyoxal–lysine dimers). A cross-link between arginine and lysine residues [MODIC (methylglyoxal-derived imidazolium cross-linking)] was also reported [131]. Quantitative analysis of AGE reveals that the hydroimidazolone MG-H1 is the major glycation adduct formed with argpyrimidine, CEL and MOLD formed as minor modifications [132134]. In human serum albumin minimally glycated by methylglyoxal, the following MAGE concentrations were detected: MG-H, 2493±87 mmol/mol protein; argpyrimidine, 200±40 mmol/mol protein; CEL, 29.7±1.8 mmol/mol protein; and MOL, 5±1 mmol/mol protein [133]. In the human lens, the AGE concentrations detected were: MG-H1, 4609±411 pmol/mg protein; MG-H2, 3085±328 pmol/mg protein; argpyrimidine, 205±19 pmol/mg protein; and pentosidine, 0.693±0.104 pmol/mg protein [132].

Figure 6 Chemical structures of MAGE

Methylglyoxal reacts preferentially with arginine side chains to form hydroimidazolones (MG-H1, MG-H2 and MG-H3), tetrahydropyrimidine (THP) and argpyrimidine, a fluorescent MAGE. Methylglyoxal reacts also with lysine residues to form CEL. Protein cross-links are also observed with lysine residues, forming MOLD, and with both lysine and arginine residues, originating methylglyoxal-derived imidazolium cross-links (MODIC). To better understand the modification caused by methylglyoxal, arginine and lysine side chains are also shown.

Glycation of other macromolecules

Besides proteins, the amino groups of nucleic acids and basic phospholipids can also be irreversibly modified by glycation reactions.

DNA is susceptible to irreversible modification by glyoxal and methylglyoxal, with deoxyguanosine being the most reactive nucleotide in physiological conditions. Several nucleotide AGEs have been described. In vivo, the major nucleotide AGEs are the imidazopurinone derivatives: GdG {3-(2′-deoxyribosyl)-6,7dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one}, from the reaction with glyoxal, and MGdG {3-(2′-deoxyribosyl)6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one}, derived from the reaction with methylglyoxal [135]. Glyoxal and methylglyoxal also reacts with deoxyguanosine, producing a stable adduct identified as CMdG (N2-carboxymethyl-deoxyguanosine) and CEdG [N2-(1-carboxyethyl)-deoxyguanosine] respectively [136,137]. Glycated DNA was detected in vivo in human samples and in human and bovine cells cultured in vitro [135,138,139]. For HL60 cells cultured in vitro, DNA glycation adducts were substantial higher (per 106 nucleotides: GdG, 2.67±0.63; MGdG, 18.2±6.5; and CEdG, 1.33±1.12) compared with 8-OxodG (8-hydroxydeoxyguanosine), the major oxidative product of DNA (per 106 nucleotides: 1.48±0.39) [135]. A similar trend was detected in peripheral human mononuclear leucocytes from healthy humans (per 106 nucleotides: GdG, 2.11±0.74; MGdG, 8.73±2.41; CEdG, 1.03±0.38; and 8-OxodG, 2.79±0.64) [135]. This indicates that glycation by methylglyoxal is a relevant type of DNA damage. There is evidence that DNA glycation causes a loss of genomic integrity associated with genotoxic effects. High methylglyoxal concentration leads to interstrand cross-links in duplex DNA [140], strand breaks [140,141] and increased mutation frequency [141143]. Transgenic embryos of diabetic mice also have an increase mutation rate linked to high glucose concentration [144]. Diseases associated with high plasma levels of dicarbonyls, such as diabetes and renal failure, are related to increased mutagenicity and cancer risk and vascular cell apoptosis [137].

Basic phospholipids (phosphatidylethanolamine and phosphatidylserine) are potential targets of the Maillard reaction, owing to the presence of free amino groups, forming lipid-linked AGEs [145]. Carboxymethylethanolamine, a lipid-linked AGE found in vivo, is considered a biomarker of phospholipid modification by the Maillard reaction [146]. This process is accompanied by oxidation of the unsaturated fatty acid side chains, with 4-hydroxyhexenal and 4-hydroxynonenal as major products [145,147]. Glycated phospholipids were detected in the rat liver, with increased levels in streptozotocin-induced diabetic animals [148]. The plasma of diabetic patients contains high levels of glycated phospholipids, identified as an Amadori product of the reaction between phosphatidylethanolamine and glucose [149]. Like proteins, amino groups from phospholipids may also react with other carbonyl-containing compounds, including methylglyoxal [150].

Glycation and disease

AGEs are dysfunctional molecular modifications that should be maintained at very low levels in living cells and there has been debate whether GLO enzymes have a detoxification role for AGEs. It has been known for a long time that glycation levels correlate with the loss-of-function and the extent of cross-linking [151]. The first longitudinal studies on the variation of the concentration of AGEs confirmed that CML and pentosidine increase with age [152] and for pentosidine a negative correlation of the accumulation rate with life span was found [153]. Similar observations were reported for CEL and MOLD, which also accumulates with age in human lens proteins [154,155]. Thus protein glycation was viewed as a post-translational modification that accumulated mostly on extracellular proteins and AGEs were slowly formed, accumulating throughout life. This is indeed true for chemically stable AGEs, such as CML, CEL and pentosidine, formed on long-lived proteins like skin collagen. However, methylglyoxal-derived glycation adducts have relatively short chemical half-lives under physiological conditions (2–6 weeks for MG-H and 2–9 days for argpyrimidine) and a slow dynamic reversibility [156]. This suggests that arginine-derived MAGEs, unlike CML and CEL, are not likely to show a time-dependent accumulation in long-lived proteins in vivo over a time scale of years where protein turnover affects the AGE accumulation rate [157]. Rather, it may reflect shorter-term periods of excessive glycation following periods of abnormal accumulation of methylglyoxal; its concentration depends on the rate of AGE formation and turnover of MAGE-modified proteins by cellular proteolysis. Moreover, protein glycation adduct residues are also formed on cellular and short-lived extracellular proteins. Therefore the protein content of MAGEs can be decreased if the concentrations of the precursor oxo-aldehydes are decreased [156]. Where MAGE accumulate with the donor age, the increase is related to a decreased activity of GLO1 and increased methylglyoxal concentration [132]. The relevance of protein glycation in physiological conditions remains under intensive research.

It was reported that arginine residues have a probability of about 20% to be located in ligand- and substrate-binding sites of proteins [158]. Therefore methylglyoxal-induced modifications may have a significant impact when glycated arginine residues are located in sites of protein–protein, enzyme–substrate and protein–DNA interaction. A high number of studies have shown that glycation has damaging effects on protein structure and biological activity. However, although some of these glycated proteins were identified in vivo, it should be appreciated that proteins are minimally modified by MG-H in biological systems and studies with proteins with high extents of MG-H modification are not physiologically relevant. Any protein with arginine and lysine residues is a target for glycation, and indeed chemical modifications by methylglyoxal and phenylglyoxal have long been used to identify the critical residues involved in enzyme catalysis [17,159]. Additionally, differences in the glycation process were observed between glycation under in vivo and in vitro conditions [160].

Nonetheless, even with a low modification extend, about 5–10% of the total cellular proteins [161], an increase in protein glycation may have relevant physiological effects. Methylglyoxal modifies human serum albumin in vivo to form mainly hydroimidazolone with minor formation of argpyrimidine, CEL and MOLD [133]. Modification of Arg410 by methylglyoxal inhibited drug binding and esterase activity owing to local structural distortion leading to the disruption of arginine-directed hydrogen bonding and the loss of electrostatic interactions [133]. We also discovered that the methylglyoxal-derived glycation of enolase, a major glycation target in yeast [58], with the formation of hydroimidazolone at Arg414 disrupts electrostatic interactions essential for dimer stability, leading to dimer dissociation and glycation-dependent activity loss [130,160].

Increased formation and accumulation of MAGE-modified proteins has been detected and implicated in disease development and progression, including diabetes and its associated vascular complications, renal failure, cirrhosis and aging (reviewed in [162]). In the context of diabetes and its related complication, several examples of the molecular effect of glycation in diabetes are known. Glucose-induced glycation of LDL (low-density lipoprotein) particles, either in the apolipoprotein B or phospholipids components, leads to a significant reduction in glycated LDL clearance (by diminishing recognition by the LDL receptor) and increases LDL susceptibility to oxidative modifications [145,163165]. Nevertheless, in these studies, LDL particles were glycated in vitro to higher extents (40% of lysine residues were modified), not detected under physiological conditions. Quantitative analysis of LDL apoliprotein B100 glycation in vivo reveals that upon examination of the fructose-lysine residue content it is not the most prevalent modification and, in fact, is not significantly increased in patients with Type 2 diabetes [166]. In contrast, the methylglyoxal-derived hydroimidazolone is the major AGE of LDL and is increased up to 4-fold in individuals with Type 2 diabetes [166]. Methylglyoxal-derived arginine modification of LDL in vivo accounts for approximately 5% of LDL content in healthy people, increasing 3–4-fold in Type 2 diabetes [166]. Methylglyoxal-modification produces small dense LDL particles with high binding affinity for arterial proteoglycan, thereby producing increased retention of LDL in the arterial wall [167].

In the vascular diseases associated with diabetes, methylglyoxal- and glyoxal-derived glycation alters the activation of the epidermal growth factor receptor, by promoting an abnormal signalling response [168]. Interestingly, endothelial cells exposed to high glucose concentrations exhibit a 70% decrease in mitogenic activity owing to glycation of the basic fibroblast growth factor [169]. Methylglyoxal also disturbs the interaction of endothelial cells with the extracellular matrix by reacting within a short collagenous region containing an RGD sequence that mediates endothelial cell adhesion to type IV collagen [170]. Disruption of these interactions contributes to the characteristic lesions found in the kidney of patients with diabetic nephropathy. Interestingly, glucose has no effect on cell adhesion, illustrating once more the relevance of methylglyoxal as the main protein glycation agent in vivo [170]. In non-diabetic healthy rats, only 5% of aortal collagen had methylglyoxal-derived MG-H adduct; this increases to 12% in diabetic rats [171]. The increased methylglyoxal-derived modification of vascular basement membrane type IV collagen at the RGD and GFOGER integrin-binding sites of collagen causes endothelial cell detachment and inhibition of angiogenesis [171].

Recently, methylglyoxal was implicated in the painful diabetic neuropathy, a medical condition for which few effective therapeutic options are available [172]. It was found that glycation of the nociceptor-specific sodium channel Nav1.8 by methylglyoxal, with the formation of hydroimidazolone, is associated with enhanced sensory neuron excitability and hyperalgesia in diabetic neuropathy.

Protein glycation has been associated with amyloid-type neurodegenerative disorders with the detection of AGE-modified proteins in amyloid deposits from Alzheimer's disease [173175], Parkinson's disease [176,177], ATTR [178,179] and dialysis-related amyloidosis [180]. A clear example of the relevance of methylglyoxal glycation in neuronal disorders is triosephosphate isomerase deficiency, a rare neurodegenerative-related disorder associated with severe multisystemic disorders [181]. Deficiency in this enzyme causes an increase in intracellular methylglyoxal concentration, which is associated with increased glycation levels and oxidative stress [181,182]. Studies in Drosophila showed that mutation of triosephosphate isomerase causes paralysis, neurodegeneration and early death [183]. This illustrates the deleterious consequences of increased flux of methylglyoxal for neuronal function and integrity, and clearly demonstrates the importance of keeping methylglyoxal-derived non-enzymatic modifications at very low levels, which relies on the detoxification role of the GLO enzymes.

In Alzheimer's disease, both extracellular Aβ-peptide amyloid plaques and intracellular neurofibrillary tangles of tau protein are highly modified with AGEs [184186], with it being suggested that protein glycation could stabilize the amyloid deposits accounting for their high insolubility and protease resistance [186]. The MAGE argpyrimidine was also detected in amyloid deposits in ATTR [179]. Recently, we showed that ATTR patients present higher levels of MAGE-modified proteins in human plasma and discovered that fibrinogen, one of the main transthyretin-interacting proteins in plasma, is a specific glycation target with an increased glycation in ATTR patients [178]. Fibrinogen has a chaperone activity able to prevent thermal-induced protein aggregation of plasma transthyretin and fibrinogen glycation may compromise its chaperone function promoting transthyretin unfolding and aggregation in ATTR [178].

Besides the direct changes in protein function by methylglyoxal modifications, AGE-modified proteins also exert cellular effects via interaction with specific AGE receptors [RAGE (receptor for AGE)] [187] that trigger an inflammatory response at the cellular level, accounting for AGE toxicity.

The generation of macromolecular post-translational modifications, resulting from the reaction with reactive aldehydes, is damaging and this ‘carbonylic stress’, an expression coined by Baynes in 1991 [188], must be compensated by proper cellular defence mechanisms.

Anti-glycation defences

Once AGEs are formed, macromolecular turnover seems to be an important clearance mechanism. Protein deglycation was described as a new form of protein repair. These enzymatic mechanisms act at the Amadori product level thereby interrupting the glycation cascade in the early steps of the Maillard reaction. Although several enzymes were found to be involved in this deglycation process [189192], no enzymatic deglycation activity was described against protein glycation by methylglyoxal. Hence, it is only natural to assign to the GLO pathway an important anti-glycation role, by converting methylglyoxal into the much less electrophilic hydroxyacid D-lactate (Figure 7). Therefore GLOs, in particular GLO1, may act as a first level of defence by removing glycating agents before the glycation process occurs [73,193]. Being able to eliminate methylglyoxal, glyoxal and other α-oxoaldehydes, GLO1 plays a critical role in preventing glycation [73]. Evidence supporting this biological function can be found in studies with yeast mutants, where an increase in protein glycation was found in GLO1-null strains [58]. In fact, the activity of this enzyme is one of the most important parameters for controlling the intracellular methylglyoxal concentration in yeast cells [194]. In endothelial cells, overexpression of GLO1 prevented the in vitro accumulation of cellular AGEs by increasing the catabolism of methylglyoxal [195]. Overexpression of GLO1 prevented the impairment of angiogenesis in hyperglycaemia [196], the impairment of NO-mediated vascular dilatation in diabetes [197] and renal ischaemia/reperfusion injury [198]. In C. elegans, it was shown that the activity of GLO1 is markedly decreased with age, which promotes the accumulation of methylglyoxal-derived adducts [199]. Overexpression of GLO1 decreases methylglyoxal-derived mitochondrial protein modifications and increases the lifespan of C. elegans [199]. Therefore GLO1 is a key enzyme of the anti-glycation defence (Figure 7).

Figure 7 The methylglyoxal crossroads

Several routes are known for methylglyoxal formation in living cells, although its main production pathway is associated to glycolysis, either non-enzymatically or through the action of methylglyoxal synthase (found only in bacteria until recently). Once formed, it readily forms adducts with thiol (glutathione, trypanothione and cysteine residues in proteins) and amino (proteins, nucleic acids and lipids) groups. Irreversible reactions with amino groups in proteins and nucleic acids lead to MAGE formation and functional impairment of these macromolecules. Coupling an irreversible enzymatic pathway that eliminates methylglyoxal, the glyoxalase system, greatly reduces glycation. The main known role of this pathway is thus as an effective glycation preventor.

However, an efficient anti-glycation mechanism is not always beneficial to cells. For example, it was demonstrated that methylglyoxal-derived glycation of α-crystallin and Hsp27, both molecular chaperones, enhance their chaperone function [200,201]. In yeast, glycation of Hsp26 and Hsp71/72 suggests that glycation causes their activation or enhances their chaperone activity [130,202]. Although this is yet to be proven in vivo, these results hint at a physiological role for methylglyoxal in the activation of chaperone proteins. Indeed, it may be advantageous to cells that some methylglyoxal is able to evade its enzymatic catabolism and glycation of specific proteins could be physiologically relevant.


The functional defence role of the glyoxalase pathway has been the rationale behind its possible exploit as a therapeutic target. GLO system inhibition could lead to an increase in the intracellular methylglyoxal concentration, hence causing cellular damage.

Tumour cells that have a high glycolytic rate are of particular interest [203,204]. Increased formation of methylglyoxal and a higher expression of the GLO enzymes are observed in such cells [205208]. However, it was shown that by increasing GLO1 expression in response to augmented levels of methylglyoxal (owing to a higher glycolytic flux), cytotoxicity is not induced and tumour cells are allowed to grow [135]. Several studies have focused on the specific inhibition of GLO1 in tumour cells and its relation to anti-proliferation and apoptosis [209211]. However, not all cancer types display a higher GLO1 activity. For example, in superficial and in invasive bladder tumours there is differential expression of the GLO enzymes: GLO2 expression is higher in the invasive tumour stage without a corresponding increase in GLO1 [212].

Other interesting organisms that prompted the investigation of new therapeutic GLO enzymes- based opportunities include protozoan parasites, in which the GLO system is quite peculiar [69]. In trypanosomatids, glutathione is functionally replaced by trypanothione [a bis(glutathione)–spermidine conjugate] [213] and the GLO pathway is no exception in thiol-dependence [67,80,95,96]. Hence, the inhibition of the parasite GLOs would lead to an increase on intracellular methylglyoxal levels that would compromise parasite viability, without affecting the host enzymes. However, other methylglyoxal catabolic pathways are present in these parasites, namely the NADPH-dependent aldose reductase, as well as methylglyoxal reductase enzymes, and the importance of the GLO pathway as a possible therapeutic target is very low (reviewed in [69]). In fact, a detailed analysis of the methylglyoxal catabolism in L. infantum revealed the robustness and the tight regulation of this system: depending on the parasite life-cycle form, GLO1 and GLO2 either increase or decrease their activities in the opposite direction of the aldose reductase activity, depending on the parasite's demand for trypanothione or NADPH [214]. The GLO pathway has also been studied as a potential drug target in P. falciparum, an apicomplexan protozoan. The remarkable features in this system include a monomeric GLO1 with two allosterically regulated active sites [66], and the presence of two GLO2 enzymes, one cytosolic and the other located at the apicoplast [83]. The potent inhibitor of GLO1, S-p-bromobenzylglutathione ethylester, showed anti-malarial activity in red blood cells infected with the parasite [215]. However, the question of whether the effect on parasite growth was owing to the inhibition of the parasite or the host enzyme was raised [83]. Other potential inhibitors have been tested against P. falciparum GLO1 (reviewed in [83]), but one must keep in mind the enzyme unique structure, with different active sites in the monomer, and its allosteric regulation.


Enzymes lost in metabolism

One hundred years ago, methylglyoxal and the GLOs were considered part of glycolysis, with the ‘intermediate’ methylglyoxal being converted into lactic acid. However, the discovery of the glutathione dependence as a co-factor for the GLOs, the non-enzymatic origin for methylglyoxal from the triose phosphates and the fact that what is formed is D- and not the L-lactate, removed methylglyoxal and the GLOs from glycolysis, leaving this system an orphan of function. So what is the purpose of this pathway? Despite the importance as a detoxifying system for oxoaldehydes, there are complementary pathways for eliminating methylglyoxal. Additionally, the assigned anti-glycation defence role has been questioned since specific proteins, namely Hsps with chaperone activity, require some glycation levels to fulfil their function.

In 1987, a role for S-D-lactoylglutathione (the product of GLO1) in the regulation of microtubule length in neutrophils, controlled by the activities of the glyoxalase enzymes, was suggested [216]. Later, it was reported that microtubule assembly was potentiated in vitro by S-D-lactoylglutathione, in the concentration range 1 μM–2 mM [217]. This enzymatic system would thus have a key role in cell metabolism, indirectly acting on the microtubular components of the cytoskeleton. However, this hypothesis was not further explored.

The product of the glyoxalase pathway, D-lactate, was considered a dead-end in metabolism, not further catabolized by cells. However, in S. cerevisiae it is a substrate for different isoforms of D-lactate dehydrogenase [218]. Also, in yeast it was recently discovered that mitochondria can uptake D-lactate, metabolize it and export newly synthesized malate through a putative D-lactate/malate antiport mechanism [219]. Whether this is a regulatory mechanism to decrease the methylglyoxal concentration from the cytosol or an alternative pathway to produce malate for gluconeogenesis is still an open question.

Is it really a pathway?

The concept of a metabolic pathway comprising GLO1 and GLO2 is a widely accepted idea. This view was first challenged in 1970 with a report of GLO2 deficiency in erythrocytes [100]. The authors analysed this deficiency in family members for three generations and demonstrated that this condition is transmitted as an autosomal recessive attribute. Moreover, they observed that leucocytes did not have the GLO2 deficiency and that the vitality of GLO2-deficient erythrocytes was not impaired [100]. In 1984, a horse without GLO2 was discovered [99] and the lack of GLO2 activity was confirmed later by kinetic studies in vivo using NMR [220]. In both of these studies there were no physiological or clinical differences related to the GLO2 deficiency.

The essentiality of these enzymes in a cell or an organism can be analysed using yeast null mutants for GLO1 and GLO2, mimicking an incomplete glyoxalase pathway. Both mutants are viable and do not show any particular phenotype when compared with the wild-type cells [58]. The double mutant Δglo1Δglo2 and the triple deletion strain Δglo1Δglo2Δglo4 were generated and no phenotype was detected [101]. The incompleteness or total absence of the GLO system occurs physiologically in some protozoan parasites [69]. One example is the trypanosomatid T. brucei that lacks GLO1 but has two GLO2-encoding genes [98], unlike other trypanosomatids such as Leishmania and T. cruzi which have both enzymes (reviewed in [69]). G. lamblia, an enteric protozoan, has two genes encoding hydroxyacylglutathione hydrolases [221], but no GLO1 gene was found [69]. As for E. histolytica, also an enteric protozoan parasite, nine genes coding for proteins that belong to the metallo-β-lactamase family were found, but without any GLO1- or GLO2-encoding genes [69]. These organisms without a complete glyoxalase pathway have alternative enzymes to fulfil the elimination of methylglyoxal, like aldo-keto reductase and methylglyoxal reductase, as mentioned above [69,222]. Yet these exceptions raise serious questions regarding the concept of a pathway composed of GLO1 and GLO2, acting sequentially, as well as its function in these organisms.

Final thoughts

It is undeniable that methylglyoxal and the accumulation of MAGE, as well as of other glycated biological molecules, have deleterious effects on living cells. Increased MAGE formation is associated with the clinical complications of diabetes and is also found in amyloid deposits that are the hallmark of several neurodegenerative diseases. As such, the glyoxalase pathway has a clear protective action on the proteome. However, micro-organisms, such as yeast, cope remarkably well with the lack of GLO1, GLO2 or both, or even the simultaneous lack of GLO1 and aldose reductase, without cell viability loss or growth effects [58,101]. The only emergent phenotype is increased glycation owing to glycolytic enzyme activity loss, although without impairment of glycolytic flux. The main reason for these unexpected results is the fact that metabolism is very robust and metabolic pathways can tolerate large variations in an individual enzyme's activity without significant flux changes, as was predicted previously [223,224]. GLO1 has a very small effect on the steady-state concentration of methylglyoxal and can be easily compensated for, either by increasing glycolytic flux or by the inhibition of TIM. TIM deficiency is linked to early death in childhood and neurological dysfunction, most likely caused by methylglyoxal and MAGE formation, despite an apparently functional GLO pathway [181]. Protozoan parasites display a remarkable diversity of GLO enzymes, some organisms being viable without one of the two GLO enzymes, the most noticeable case being T. brucei that lacks GLO1 and the ability to eliminate methylglyoxal through this pathway (reviewed in [69]). Moreover, GLO enzymes from trypanosomatids are trypanothione-dependent, providing important molecular models of glutathione/trypanothione substrate specificity. Amyloid-type neurodegenerative disorders also offer a rich research ground for GLO, methylglyoxal and glycation. Since amyloid deposits contain glycated proteins, considerable research has been devoted to the effect of glycation on protein structure, unfolding pathways, aggregation and amyloid deposit formation. However, the effects of glycation are likely to be more subtle and related to energy catabolism and proteostasis. GLO3 may be highly relevant in these cases as the human equivalent was found to be DJ-1 [117]. Mutations in DJ-1 are linked to an early onset of Parkinson's disease and it remains to be seen whether these mutants have impaired glyoxalase activity and are associated with MAGE accumulation. Increased glycation has also been found in ATTR patients [178] and in a mouse knockout lacking synuclein [225], the main component of Lewy bodies in Parkinson's disease. In both cases, the ultimate causes for the increase in glycation are not known, but seem to be far from harmless.

GLO research has experienced many twists and turns since its discovery 100 years ago, as a function of the perceived relevance of these enzymes for metabolism and human health. Glycation, methylglyoxal and GLO might well be a missing link in conformational neurodegenerative diseases, whether as a pathway or not. Research in this area is likely to bring GLO to the limelight once more.


Work in the author's laboratory is supported by projects PTDC/SAU-MIC/115178/2009 and PEst-OE/QUI/UI0612/2011 and the Fundação para a Ciência e a Tecnologia, Portugal [grant number SFRH/BPD/41037/2007 (to R.A.G.)].


We wish to thank Paul John Thornalley for bringing the focus on methylglyoxal and the GLO pathway after a long oblivion and for all his foresight on glycation research, as well as lively and thought-provoking discussions.

Abbreviations: AGE, advanced glycation end-product; ATTR, transthyretin amyloidosis; CEdG, N2-(1-carboxyethyl)-deoxyguanosine; CEL, Nϵ-(carboxyethyl)lysine; CML, N-(carboxymethyl)lysine; DHAP, dihydroxyacetone phosphate; GAP, D-glyceraldehyde 3-phosphate; GdG, 3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one; GLO, glyoxalase; Hsp, heat-shock protein; LDL, low-density lipoprotein; MAGE, methylglyoxal-derived AGE; MGdG, 3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one; MOLD, methylglyoxal–lysine dimer; 8-OxodG, 8-hydroxydeoxyguanosine; TIM, triose phosphate isomerase


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