Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of 1 H nuclear magnetic resonance ( 1 H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with 1 H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.Methylglyoxal (MG) is a widely occurring ketoaldehyde that is accumulated under physiological conditions with uncontrolled carbohydrate metabolism (1,11,20). MG synthesis is mediated by enzymes, including methylglyoxal synthase, cytochrome P450, and amine oxidase, which are involved in glycolytic bypass, acetone metabolism, and amino acid breakdown, respectively (8,18). In eukaryotic cells, MG is also generated by nonenzymatic fragmentation of dihydroxyacetone phosphate or glyceraldehyde 3-phophate (28). MG is a highly toxic electrophile and reacts with cellular macromolecules, including DNA and proteins (16,18).There are various ways that the cellular degradation of MG occurs (Fig. 1). The glyoxalase system, consisting of glyoxalase I and II, converts MG into D-lactate in the presence of glutathione (30). The conversion of MG into lactaldehyde by the MG reductase was also suggested (26, 29). The enzymes, presumably aldose and aldehyde reductases, mediating the reduction of MG to acetol and D-lactaldehyde have been reported for Escherichia coli, yeast (Saccharomyces cerevisiae), plants, and mammals (16,25,31). The mammalian aldo-keto reductase (AKR) family AKR1, AKR1A1 (EC 1.1.1.2), and AKR1B1 (EC 1.1.1.21) and the family AKR7, AKR7A2, and AKR7A5 convert methylglyoxal to acetol in the presence of NADPH (14,15,27,31,32). The E. coli YghZ protein, belonging to the AKR14 family, was recently characterized as an enzyme involved in MG reduction and was also shown to enhance resistance to MG when overproduced (12).Aldo-keto reductases encompass a large superfamily of NADPH-dependent oxidoreductases that reduce various aldehydes and ketones (17). They all share a common (␣/␤) 8 -barrel motif characteristic of triose phosphate isomerase. Most AKRs are monomeric, with the exce...

By exploiting nuclear magnetic resonance (NMR) techniques along with novel applications of saturation difference analysis, we deciphered the functions of the previously uncharacterized products of three bacterial genes, rbsD, fucU, and yiiL, which are part of the ribose, fucose, and rhamnose operons of Escherichia coli, respectively. We show that RbsD catalyzes the pyran to furan conversion of ribose, whereas FucU and YiiL are involved in the catalysis of the anomeric conversion of their respective sugars. It was observed that the anomeric exchange of only ribofuranose, not ribopyranose, occurs spontaneously in solution rationalizing its evolutionary incorporation into the nucleic acid. The RbsD and FucU proteins share sequence homology and belong to the same protein family that is found from eubacteria to human, whereas the YiiL homologues exist in archaebacteria and lower eukaryotes. These enzymes, including the galactose mutarotase, exhibit a certain degree of cross-specificity to structurally analogous sugars thereby encompassing all existing monosaccharides in terms of their reactivities. The ubiquitous presence of enzymes involved in the anomeric changes of monosaccharides highlights an importance of these activities in various cellular processes requiring efficient monosaccharide utilization.The ␣-␤ anomeric change of a monosaccharide involving a pyran configuration is considered very slow, e.g. the rate of glucose mutarotation is 0.015 min Ϫ1 in water (1). The only known enzyme for such a process is galactose mutarotase (GalM of Escherichia coli), which accelerates the conversion between the ␣-and ␤-anomers of D-glucose and D-galactose (2-4) presumably by enhancing the efficiency of glycolysis. The enzymes involving sugar metabolism including glycosylation tend to be selective for the ␣-or ␤-anomer, and thus a slow anomeric conversion may cause a problem in utilizing sugar. There has been no report on proteins involving an ␣-␤ conversion of sugars other than glucose, perhaps because of the lack of a method for detecting mutarotation at equilibrium. The conventional methods can only detect a change in optical rotation caused by an equilibrium shift from either the ␣-or ␤-anomer, which requires a purified ␣-or ␤-anomer as a substrate (3).Monosaccharides in general have ␣-and ␤-anomers of pyranose (six-membered ring, e.g. L-fucose and L-rhamnose) and often have additional ␣-and ␤-anomers of furanose (five-membered ring). D-Ribose, a key sugar moiety for the component of nucleic acids and ATP, exists in four different forms in solution, ␣-pyranose (22%), ␤-pyranose (58%), ␣-furanose (7%), and ␤-furanose (12%), with its open-chain form constituting less than 1% of the total ribose in solution. Ribose is transported as a ␤-D-pyranoribose, which is the major form in solution and binds to the ribose-binding protein (RbsB) in the bacterial periplasm (5). However, the ribokinase (RbsK) involved in the next step of ribose metabolism recognizes ␣-D-ribofuranose as a substrate (6). An apparent discrepancy in the subs...

Methylglyoxal (MG) is a highly reactive metabolic intermediate, presumably accumulated during uncontrolled carbohydrate metabolism. The major source of MG is dihydroxyacetone phosphate, which is catalyzed by MG synthase (the mgs product) in bacteria. We observed Escherichia coli cell death when the ribose transport system, consisting of the RbsDACBK proteins, was overproduced on multicopy plasmids. Almost 100% of cell death occurs a few hours after ribose addition (>10 mM), due to an accumulation of extracellular MG as detected by 1 H-nuclear magnetic resonance ( 1 H-NMR). Under lethal conditions, the concentration of MG produced in the medium reached approximately 1 mM after 4 h of ribose addition as measured by high-performance liquid chromatography. An excess of the protein RbsD, recently characterized as a mutarotase that catalyzes the conversion between the ␤-pyran and ␤-furan forms of ribose, was critical in accumulating the lethal level of MG, which was also shown to require ribokinase (RbsK). The intracellular level of ribose 5-phosphate increased with the presence of the protein RbsD, as determined by 31 P-NMR. As expected, a mutation in the methylglyoxal synthase gene (mgs) abolished the production of MG. These results indicate that the enhanced ribose uptake and incorporation lead to an accumulation of MG, perhaps occurring via the pentose-phosphate pathway and via glycolysis with the intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate. It was also demonstrated that a small amount of MG is synthesized by monoamine oxidase.