Metabolism of 2-oxoaldehyde in yeasts. Purification and characterization of NADPH-dependent methylglyoxal-reducing enzyme from Saccharomyces cerevisiae

Abstract

An enzyme catalyzing the reduction of methylglyoxal was isolated from Saccharomyces wrevisiae and its enzymatic properties were analyzed. The enzyme, specifically eluted from a blue-dextran -Sepharose CL-6B column by the substrate, methylglyoxal, was homogeneous on polyacrylamide gel electrophoresis. The enzyme consisted of single polypeptide chain with a relative molecular mass of 43000. The enzyme was glycoprotein and contained 6.6% carbohydrate. NADPH was specifically required for activity and the K, for NADPH was 2.0 x lo-' M. The enzyme was active on various glyoxals such as glyoxal, methylglyoxal ( K , = 5.88 mM) and phenylglyoxal ( K , = 1.54 mM). The reaction catalyzed by the enzyme was virtually irreversible. The aclivity was inhibited by sulfhydryl agents and activated by reducing agents such as glutathione. Intermediates in glycolysis, nucleosides, nucleotides, polyamines and various metal ions showed little inhibitory or activating effects on enzyme activity. Tricarboxylic acids showed a slight inhibitory effect. The activity of the enzyme was strongly inhibited by anionic detergents. The enzyme was rapidly inactivated by incubating with the substrates probably because of the non-enzymatic interaction between glyoxals and NH2 groups in arginine residues in the enzyme. NADP, one of the reaction products, also inhibited the enzyme activity and the Ki for NADP was about 0.07 mM.We tentatively designated the enzyme methylglyoxal reductase.

Purification and Characterization ofα-Keto Ester Reductases from Bakers’ Yeast

“…Kinetic measurements have shown that YKER-IV only is effective for the reduction of ex-keto esters at low substrate concentrations. The MichaelisMenten constant of YKER-IV is explicitly smaller than those of other two enzymes, YKER-II and -V. YKER-IV showed similar properties to those of methylglyoxal reductase 16 ) and ketopantoyllactone reductase 13 15) from the viewpoint of substrate specificity and molecular mass of these enzymes. Both enzymes were isolated from Saccharomyces cerevisiae.…”

“…Molecular mass of methyl glyoxal red uctase is reported to be 25.7 kDa by Sephadex 0-150 gel filtration and 43 kDa by SDS-PAOE. 16 ) The molecular mass of ketopantoyllactone reductase has been estimated to be 27.4 kDa for Form A and 27 kDa for Form B based on the results from gel filtration.14) Catalytic activities for the reductions of methylglyoxal, phenylglyoxal, and ketopantolactone by YKER -IV are very large compared to those for ex-keto esters. Kms ofYKER-IV for phenylglyoxal and methyl glyoxal were 0.651 mM and 1.93 mM, respectively and these Kms are similar to those of methyl glyoxal reductase for phenylglyoxal (1.54mM) and methyl glyoxal (5.88mM).…”

“…ll ,12) Only a few enzymes that catalyze the reduction of keto esters other than f3-keto esters have been isolated; these are ketopantolactone reductase, ketopantoic acid reductase, 13 15) and methyl glyoxal reductase. 16 ) (X-Keto ester reductases have not been isolated from bakers' yeast or other microbes except for glycerol dehydrogenase from Geotrichum candidum. 17 ,18) It is necessary to isolate and purify (X-keto ester reductases (YKERs) to understand the mechanism of stereochemical control of reduction by yeast.…”

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cellsin vitro

“…MG reductase activity was determined spectrophotometrically as described [31] with minor modification: using 1 mM MG as substrate and 50 mM sodium phosphate buffer, pH 7.4 at 37°C, as the assay buffer. D-Lactate was measured by endpoint enzymatic assay with microplate fluorimetry [32].…”

Metabolism of 2-oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in l-threonine catabolism by Saccharomyces cerevisiae

“…To elucidate the function of methylglyoxal in cell proliferation, we have examined the metabolic fate of methylglyoxal in yeast Saccharomyces cerevisiae cells and found an alternative route for methylglyoxal degradation besides the glyoxalase system consisting of glyoxalase I and glyoxalase 11. In this route, methylglyoxal is converted into L-lactaldehyde by NADPH-dependent methylglyoxal reductase [6] and L-lactaldehyde is then converted to L-lactate by NAD-dependent L-lactaldehyde dehydrogenase [7]. The NADPH-dependent methylglyoxal reductase has been found in all the microbial (Escherichia, Bacillus, Pseudomonas species, Actinomycetes and molds) and mammalian (brain, liver, heart, spleen and kidney of rat) cells at a considerably high activity and was thought to have a significant role in methylglyoxal metabolism in combination with the glyoxalase system (unpublished results).…”