Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos

Abstract

The sugar-induced inhibition of malolactic fermentation in cell suspensions of Leuconostoc oenos, recently reclassified as Oenococcus oeni (L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995) was investigated by in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and manometric techniques. At 2 mM, glucose inhibited malolactic fermentation by 50%, and at 5 mM or higher it caused a maximum inhibitory effect of ca. 70%. Galactose, trehalose, maltose, and mannose caused inhibitory effects similar to that observed with glucose, but ribose and 2-deoxyglucose did not affect the rate of malolactic activity. The addition of fructose or citrate completely relieved the glucose-induced inhibition. Glucose was not catabolized by permeabilized cells, and inhibition of malolactic fermentation was not observed under these conditions. 31 P NMR analysis of perchloric acid extracts of cells obtained during glucose-malate cometabolism showed high intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate. Glucose-6-phosphate, 6-phosphogluconate, and NAD(P)H inhibited the malolactic activity in permeabilized cells or cell extracts, whereas NADP ؉ had no inhibitory effect. The purified malolactic enzyme was strongly inhibited by NADH, whereas all the other above-mentioned metabolites exerted no inhibitory effect, showing that NADH was responsible for the inhibition of malolactic activity in vivo. The concentration of NADH required to inhibit the activity of the malolactic enzyme by 50% was ca. 25 M. The data provide a coherent biochemical basis to understand the glucose-induced inhibition of malolactic fermentation in L. oenos.Malolactic fermentation is a process that occurs in wine after alcoholic fermentation and consists of the conversion of Lmalate to L-lactate and carbon dioxide. As a consequence of this reaction, the total acidity decreases and the organoleptic properties and biological stability of the wine are generally improved (10,11,41). Several studies have shown that Leuconostoc oenos is well adapted to high ethanol concentrations and low pH values and is largely responsible for malolactic fermentation in wine (8,15,18,20). Although L. oenos was recently reclassified as Oenococcus oeni (13), the old designation will be used throughout this work. The malate fermentation pathway in L. oenos generates a proton motive force that drives ATP synthesis (9, 29), thus explaining the early report of a pH-independent stimulation of growth by malate (24). Glucose and fructose, the major sugars present in wine, can be utilized by L. oenos as energy sources for growth (41), and another important component in wine, citric acid, also plays an important role in the bioenergetics of this bacterium (26).The malolactic reaction is catalyzed by the malolactic enzyme, which has been purified from several organisms (2, 4, 22). The malolactic enzymes studied so far are homodimers or tetramers of 60-to 70-kDa subunits, have a K m for malate ranging fro...

Taxonomic Status of Lactic Acid Bacteria in Wine and Key Characteristics to Differentiate Species

“…MLF is regulated by glucose. As little as 2 mM glucose inhibits MLF by 50% (Miranda et al, 1997). Similar results were recorded in the presence of galactose, trehalose, and maltose.…”

“…Similar results were recorded in the presence of galactose, trehalose, and maltose. Ribose and 2-deoxyglucose, however, had no effect on the conversion of L-malic acid (Miranda et al, 1997). Glucose inhibition is reversed by the addition of fructose or citrate, which leads to an increase in intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate (Miranda et al, 1997).…”

“…Ribose and 2-deoxyglucose, however, had no effect on the conversion of L-malic acid (Miranda et al, 1997). Glucose inhibition is reversed by the addition of fructose or citrate, which leads to an increase in intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate (Miranda et al, 1997). Phenolic compounds in red wine reduces the rate of sugar consumption by O. oeni, but enhances the metabolism of citric acid, which in turn leads to increased levels of acetic acid (Rozés et al, 2003).…”

Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni

“…ND, not detected; MDH, metal-dependent hydrolase; GR, glutathione reductase; GPDH, glyceraldehyde-3-phosphate dehydrogenase; IMPDH, inosine-5Ј-monophosphate dehydrogenase; HPr, phosphocarrier protein. (30). Moreover, phosphomethylpyrimidine (HMP-P) kinase, which catalyzes the stepwise phosphorylation of HMP-P into HMP-PP (21), a heterocyclic intermediate in the de novo synthesis of thiamine diphosphate (ThDP), was increased in ethanol-adapted cells.…”

Flow Cytometric Assessment of Membrane Integrity of Ethanol-StressedOenococcus oeniCells

“…This hypothesis is supported by the fact that cF leakage followed the global trend of ethanol concentration dependence that paralleled the increase in passive proton influx rates at increasing ethanol concentrations. NAD ϩ is an important cofactor of the malolactic enzyme, and it has been reported that the delicate balance between NAD ϩ and NADH affects the activity of the enzyme (28). Conceivably, nonspecific leakage of intracellular material will significantly disturb this balance, and consequently it is expected that cell metabolism will be negatively affected.…”