On 27–29 November 2013, researchers gathered at the University of Warwick, Coventry, U.K., to celebrate the centennial of the discovery of the glyoxalase pathway. The glyoxalase system was discovered and reported in papers by Carl Neuberg and by Henry Drysdale Dakin and Harold Ward Dudley in 1913. All three were leading extraordinary investigators in the pioneering years of biochemistry. Neuberg proposed glyoxalase as the pathway of mainstream glycolysis and Gustav Embden correctly discounted this, later confirmed by Otto Meyerhof. Albert Szent-Györgyi proposed glyoxalase I as the regulator of cell growth and others discounted this. In the meantime, molecular, structural and mechanistic properties of the enzymatic components of the system, glyoxalase I and glyoxalase II, have been characterized. The physiological function of the glyoxalase pathway of enzymatic defence against dicarbonyl glycation, particularly by endogenous methylglyoxal, now seems secure. We are now in an era of investigation of the regulation of the glyoxalase system where a role in aging and disease, physiological stress and drug resistance and development of healthier foods and new pharmaceuticals is emerging. The history of glyoxalase research illustrates the scientific process of hypothesis proposal, testing and rejection or acceptance with further investigation, standing testament to the need for intuition guided by experience and expertise, as well as indefatigable experimentation.
“If I have seen further it is by standing on the shoulders of giants” Isaac Newton
Early pioneers of glyoxalase research
Carl Neuberg was born in Hanover in 1877. He obtained his Ph.D. in 1900 under the supervision of Alfred Wohl in the Institute of Chemistry at the University of Berlin. Alfred Wohl was a specialist in carbohydrate chemistry and directed Neuberg's interest to the chemistry of organic compounds containing three carbon atoms. Part of Neuberg's Ph.D. thesis dealt with glyceraldehyde and purification of the derived osazone, the adduct formed from saccharide-derived dicarbonyls with phenylhydrazine. He became a lecturer at the University of Berlin in 1903, professor in 1906 and institute director in 1909 of the Animal Physiology section of the Agricultural College in Berlin. In 1913, Neuberg became head of the newly founded Kaiser Wilhelm Institute for Experimental Therapy in Berlin. Neuberg found that animal tissues converted methylglyoxal into lactic acid and called this activity glyoxalase . He developed his first fermentation diagram, the Neuberg schema. This proposed five steps in the conversion of glucose into alcohol in which methylglyoxal was an intermediate .
Henry Drysdale Dakin (1880–1952) was born in London and studied chemistry at the University of Leeds supervised by Julius Berend Cohen, gaining a Ph.D. working on amino acids. He then worked with Albrecht Kossel on arginase at the University of Heidelberg, Germany. He joined Columbia University, New York, NY, U.S.A., in 1905 and worked in the private laboratory of Christian Herter (1865–1910), a physician and pathologist, built in the fourth floor of his house on 819 Madison Avenue. He worked on fundamental research in biochemistry, and remained in charge of the laboratory after Herter's early death in 1910. Harold Ward Dudley was born in Derby, England, in 1887. He studied chemistry at the University of Leeds and was known to Julius Cohen. He went to the Institute of Chemistry at the University of Berlin and gained his Ph.D. there in 1912 under the supervision of Wilhelm Traube. In 1912, Dakin was seeking an assistant and collaborator, and, through his old teacher, Cohen was brought into touch with Dudley who was offered and took up a 2-year appointment. Dakin and Dudley published their glyoxalase discovery paper on administration of phenylglyoxal to rabbits and found the related 2-hydroxyacid, mandelic acid, was formed. They noted the possible relevance for the metabolism of methylglyoxal and added a note in proof that “further investigation has shown that the enzyme above referred to is active in aqueous extracts of various tissues and that it may be roughly purified by precipitation with salts. An enzyme solution prepared from dog's liver when added to pure methyl glyoxal (4 grams), prepared according to Meisenheimer's method, effected its complete decomposition in less than ten minutes with formation of lactic acid” . They published nine further papers on glyoxalase and related studies.
Rise and fall of glyoxalase in mainstream glycolysis
In the late 1920s, Neuberg devoted himself to strengthen the role of methylgloxal and glyoxalase in mainstream glycolysis, supporting his view by experiments showing that 80–100% of methylglyoxal added to bacteria was converted into lactic acid, and asserted the view that “the formation of methylglyoxal is the essence of glycolysis” .
Gustav Georg Embden (1874–1933) was born in Hamburg and studied chemistry. He taught at the University of Frankfurt from 1914 and conducted studies on carbohydrate metabolism and muscle contraction. Embden opposed Neuberg's view that methylglyoxal was central to glycolysis because he found that glycolysis produced only the L-enantiomer of lactic acid, whereas, with addition of methylglyoxal to tissues, he found both L- and D-lactic acid. Embden found that F-1,6-BP (fructose 1,6-bisphosphate) was converted into 3-phosphoglycerate. He concluded that 3-phosphoglycerate was the oxidation product of GA3P (glyceraldehyde 3-phosphate) and this must have been formed by splitting F-1,6-BP into two triosephosphates, GA3P and DHAP (dihydroxyacetone phosphate). He also proposed that 3-phosphoglycerate was a precursor of pyruvate and L-lactate . He wrote, “as one can see, this picture of glycolysis omits the methylglyoxal that was accepted by Neuberg as the intermediary product in yeast fermentation as well as in glycolytic lactate formation”. The long reign of Neuberg's views was over . The role of inorganic phosphate was resolved by Warburg, finding that GA3P oxidized to 1,3-bisphosphoglycerate .
Otto Meyerhof, born in Hanover in 1884, gained an M.D. at the University of Heidelberg in 1909 and shared the 1922 Nobel Prize in Medicine with A.V. Hill for this work on the energy changes in cellular respiration, including the role of lactic acid. His work, after the death of Embden in 1933, confirmed in experimental investigation the intuitive proposal of glycolytic intermediates by Embden  and together they are credited with discovering the Embden–Meyerhof pathway, the pathway involved in the conversion of glucose into L-lactate.
Emergence of characteristics and physiological function of the glyoxalase system
In the 1930s–1950s, advances were made that characterized the essential aspects of the glyoxalase system that we know today. Karl Lohmann (1898–1978) discovered that glutathione (GSH) was a specific and essential cofactor for methylglyoxal metabolism by the glyoxalase system . Maurice Jowett and Juda Quastel presented evidence that GSH and methylglyoxal combined reversibly to form a hemithioacetal substrate of the glyoxalase system . Samuro Yamazoye found that the hemithioacetal was converted into a novel acid-stable base-labile intermediate , now known to be S-D-lactoylglutathione. Frederick Gowland-Hopkins and Edward J. Morgan found wide distribution of the glyoxalase in living organisms . Efraim Racker discovered that the glyoxalase system consisted of two enzymes acting in sequential metabolic steps: Glo1 (glyoxalase I), which catalysed the formation of S-lactoylglutathione from GSH and methylglyoxal, and Glo2 (glyoxalase II) which hydrolysed S-lactoylglutathione to lactate and reformed GSH . Racker indicated that D-lactate was the product of the glyoxalase system in crude or partially purified systems in 1954 . This was verified later by Ekwall and Mannervik who analysed the enantiomer composition of hydrolysis products of S-D-lactoylglutathione prepared from enzymatic synthesis with purified Glo1 and found D-lactate . This established the core characteristics of the glyoxalase system (Figure 1).
The glyoxalase system and formation of methylglyoxal from the Embden–Meyerhof pathway
With the role of methylglyoxal and glyoxalase in glycolysis discounted, investigators speculated on the functions of the glyoxalase system. Albert Szent-Györgyi, recipient of a Nobel Prize for the discovery of vitamin C, proposed that the conflict of methylglyoxal and Glo1 controlled cell growth and could be exploited for treatment of cancer. Methylglyoxal was hypothesized to be a growth-retarding substance or ‘retine’ and Glo1 a counter to this growth restriction effect or ‘promine’ . The discovery of many other growth factors and change of cell responsiveness to them on malignant transformation led to the redundancy and demise of this hypothesis. High concentrations of methylglyoxal were toxic to tumour cells and Vince and Wadd  suggested that Glo1 inhibitors may be more effective anticancer agents, producing toxicity through accumulation of endogenous methylglyoxal.
In the 1970s–1990s, glyoxalase enzymes were purified, and molecular, kinetic, mechanistic and structural characteristics were identified. Distinctive molecular characteristics of mitochondrial matrix and cytosolic Glo2 were also identified, originating from one gene in mammals by mRNA splicing. This culminated with catalytic mechanisms of action of Glo1 and Glo2 and crystal structures of human Glo1 and Glo2. Mannervik and co-workers made a leading contribution for microbial and mammalian Glo1, with contributions of others, notably, Kimura, Norton, Principato, Thornalley, Uotila and Vander Jagt. Examples of key studies are [18–26].
An outstanding problem was the physiological substrate of the glyoxalase system. In 1933, Lohmann and Meyerhof  had found that phosphate was eliminated from triosephosphate when refluxed at 100°C in 1 M HCl or at ambient temperature in 1 M sodium hydroxide. Studies with DHAP with yeast and bacterial extracts suggested the presence of an enzyme, methylglyoxal synthase, converting DHAP into methylglyoxal [28,29]. Linked to glyoxalase and microbial D-lactic dehydrogenase, this was proposed as a bypass for the DHAP to pyruvate stage of the Embden–Meyerhof pathway. Circumspect interpretation is required, however, as high pH sample processing was often used which promotes the conversion of DHAP into methylglyoxal non-enzymatically. The problem was finally resolved by application of a specific assay for methylglyoxal to studies of the spontaneous and enzymatic formation of methylglyoxal from GA3P and DHAP under physiological conditions. Methylglyoxal formation from triosephosphates was spontaneous and unavoidable in mammalian cells, ‘leaking’ from the Embden–Meyerhof pathway, and, although occurring at a low level, the high reactivity of methylglyoxal with protein and DNA required an efficient route for methylglyoxal detoxification provided by the glyoxalase pathway .
Physiological functions proposed for the glyoxalase system have been: control of cell growth , glycolytic bypass in microbial systems , defence against dicarbonyls absorbed from gastrointestinal bacteria , regulation of microtubule assembly , and enzymatic defence against dicarbonyl glycation . The last function is the one that now seems secure. The timeline of historical development in glyoxalase research is summarized in Figure 2.
Timeline of key discoveries in glyoxalase research in the last 100 years
Glyoxalase in health and disease
In recent years, glyoxalase research has seen marked expansion in studies of disease, aging, therapeutics and environmental stress resistance.
The glyoxalase system has a longstanding association with cancer research. Although potent glutathione-derived substrate analogue inhibitors of Glo1 were developed as prospective anticancer agents from 1969, antitumour activity was not achieved. In 1992, Thornalley considered that this might be due to poor membrane permeability and extracellular instability of the inhibitors and produced the first cell-permeant Glo1 inhibitor, S-p-bromobenzylglutathione diester, with antitumour activity in vitro and in vivo [34,35], which were developed further by Creighton and co-workers . Tsuruo and colleagues discovered a link of overexpression of Glo1 to MDR (multidrug resistance) in cancer chemotherapy and sensitivity of tumours with high Glo1 expression to cell-permeant Glo1 inhibitors . Santarius et al.  found increased GLO1 copy number in drug naïve human tumours, particularly tumours of breast and lung, suggesting that some human tumours have innate MDR for which Glo1 inhibitor drugs may provide improved treatment.
In 1988, Thornalley discovered increased formation of methylglyoxal by cultured cells in high glucose concentration, a model for hyperglycaemia in diabetes  and later a link between methylglyoxal and Glo1 to the development of microvascular complications of diabetes (nephropathy, retinopathy and neuropathy) . This has recently been consolidated in mechanistic studies linking neuropathy to modification of neuronal voltage-dependent sodium channels Nav1.8  and functional genomic studies of Glo1 linking protein glycation by methylglyoxal to diabetic nephropathy . A genetic link of Glo1 to obesity was identified in 1991 , but its significance is not yet understood.
In other research, there have been developments of glyoxalase involvement in pathological anxiety , schizophrenia , Parkinson's disease , Alzheimer's disease , parasitology  and environmental stress resistance in plants . Overexpression of Glo1 in the nematode Caenorhabditis elegans produced an approximately 30% increase in lifespan, and Glo1 silencing produced a 40% decrease in lifespan, unequivocally linking Glo1 and glycation by methylglyoxal to longevity .
In the future, we are likely to see further research on glyoxalase involvement in development and progression of metabolic, vascular, neurological and degenerative disease and aging; metabolic and inflammatory regulation of glyoxalases; development of Glo1 inducer functional foods and therapeutic agents; and development of Glo1 inhibitors for MDR tumours and pathogenic micro-organisms. Glyoxalase and related methylglyoxal-modified proteins, peptides and free adducts may provide biomarkers for clinical diagnostics; and GLO1 copy number variation in plant species may provide crops endogenously resistant to environmental stress. As Isaac Newton said when commenting on the process of discovery, “I keep the subject constantly before me…”.
A commemorative video on the history of glyoxalase research relating to this paper with a compilation of recollections, reflections and prospective views by glyoxalase researchers was shown at the conference dinner and has been given to the Biochemical Society for their historical archive.
Glyoxalase Centennial: 100 Years of Glyoxalase Research and Emergence of Dicarbonyl Stress: A Biochemical Society Focused Meeting held at the University of Warwick, U.K., 27–29 November 2013. Organized and Edited by Naila Rabbani and Paul Thornalley (University of Warwick, U.K.).
We thank all speakers, poster presenters, other participants and sponsors of the Glyoxalase Centennial conference.