Metabolism of 2‐oxoaldehydes in yeasts

Abstract

NAD-dependent lactaldehyde dehydrogenase, catalyzing an oxidation of lactaldehyde to lactate, was purified approximately 70-fold from cell extracts of Saccharomyces cerevisiae with a 28% yield of activity. The enzyme was homogeneous on polyacrylamidc gel electrophoresis. The relative molecular mass of the enzyme was estimated to be 40000 on Sephadex G-150 column chromatography and on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5, 60 -C and specifically oxidized L-lactaldehyde to L-lactate in the presence of NAD. The K, values for L-lactaldehyde and NAD were 10 mM and 2.9 mM, respectively. The purest enzyme was extremely unstable and almost completely inactivated during storage at -20 C, pH 7.5. For the reactivation of the enzyme, halide ions such as CI-, I and Br-were required.The metabolism of lactaldehyde in yeast cells has been studied by Neuberg and Vercellone [I] and then by Sandman and Miller [2]. Their investigations were designcd to study only the metabolic relationship between lactaldehyde, acetol (1 -hydroxy-2-propanone), 1,2-propane-diol and lactate. The data obtained by them indicated that the lactaldehyde was converted to 1 ,2-propane-diol, lactate and acetol, thc acetol probably being derived from 1,2-propane-dioI [ 11. However, no data were given for the nature of the enzymes involved in the metabolism of these compounds.Recently, we found that yeast cells of Saccharomyces wrrvisiae could convert methylglyoxal to lactaldehyde by the action of NADPH-dependent methylglyoxal reductase and the reaction catalyzed by the reductase was virtually irreversible [3]. Further enzymatic oxidation of lactaldehyde is, therefore, considered to result in either of two products, 1,2-propane-diol and lactate. To elucidate the fate of lactaldehyde formed from methylglyoxal, the enzyme catalyzing the oxidation of lactaldehyde to lactate was sought, purified and its properties were characterized. MATERIALS AND METHODS Chemicalswere cultured in a nutrient medium (1.0% yeast extract, 2.0% peptone, 2.0% glucose, pH 5.0; 1.5 1 medium in a 2-1 Sakaguchi flask) at 30°C for 24 h with reciprocation. Cells (55 g as wet weight from 10 1 of culture) after centrifugation were washed once with 0.85% saline solution, resuspended in 10 mM Tris/HCI buffer (pH 7.5) containing 0.1 mM methylphenylsulfonyl fluoride (PhMeS02F) [Tris/ PhMeSO,F]. The cell suspension was homogenized on a Dyno-Mill for 4 min at 0' C. The supernatant after centrifugation at 8000 rpm was used for the purification of lactaldehyde dehydrogenase. Assa,yfor lactaldehyde dehydrogenaseThe reaction mixture (3.0 ml) contained 4.3 mM L-lactaldehyde, 5.0 mM NAD, 50 mM Tris/HCl buffer (pH 6.5) and enzyme. The reaction was initiated by the addition of enzyme and an increase in absorbance at 340 nm at 25°C was recorded on a Hitachi spectrophotometer model 100-50. Lactaldehyde concentration was determined by bisulfate uptake [5]. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 .O...

Metabolism of 2‐oxoaldehyde in mold

“…Although voluminous enzymatic and physiological studies have been done on the glyoxalase system, the significance of the reduction/oxidation system for the [ll], and from mammals (goat liver) [lo]. Lactaldehyde dehydrogenase has been purified from S. cerevisiue [14] and Escherichiu coli [15]. To ascertain the function of a reduction/oxidation system for the detoxification of methylglyoxal, we isolated two kinds of methylglyoxalreducing enzymes from Aspergillus niger.…”

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

“…The glyoxalase system (glyoxalase I and glyoxalase 11) has been considered to have a ubiquitous function in a detoxification of methylglyoxal. However, we have recently found another methylglyoxal detoxification route consisting of NADPH-linked methylglyoxal reductase [6] and NADlinked L-lactaldehyhde dehydrogenase [7]. This route catalyzes the conversion of methylglyoxal to L-lactate via Llactaldehyde and builds up a glycolytic bypath together with glyoxalase system as shown in Fig.…”

“…The yeast cells used contained no other methylglyoxaldegrading enzymes other than the two routes shown in Fig. 4 and in our previous paper [7]. Since methylglyoxal and other 2-oxoaldehydes are known to be carcinostatic [23] and S-lactoylglutathione has been suggested to be a growth stimulator [24], the regulation factor determining the flow of methylglyoxal to lactate via S-lactoylglutathione or L-lactaldehyde should be clarified to ascertain the function of the glycolytic bypath in yeast cell proliferation.…”

“…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).…”

Metabolism of 2-Oxoaldehydes in Bacteria: Purification and Characterization of Methylglyoxal Reductase fromEscherichia coli

“…The procedures for the purification of lactaldehyde dehydrogenase are described in our previous paper. 2 ) -e-, methylglyoxal; -0-, lactaldehyde; -0-,…”