scholarly journals YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis

2006 ◽  
Vol 188 (13) ◽  
pp. 4727-4736 ◽  
Author(s):  
Guillaume Lerondel ◽  
Thierry Doan ◽  
Nicola Zamboni ◽  
Uwe Sauer ◽  
Stéphane Aymerich
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Malic Enzyme ◽  
Metabolic Flux ◽  
Physiological Role ◽  
Extreme Case ◽  
Growth Defect ◽  
Flux Analysis ◽  
Malic Enzyme Activity

ABSTRACT The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD+ than with NADP+. YtsJ activity, in contrast, was 70-fold higher with NADP+ than with NAD+, with Km values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

scholarly journals Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Manuel Hörl ◽  
Tobias Fuhrer ◽  
Nicola Zamboni
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Content Type ◽  
Malolactic Enzyme ◽  
First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme activities by H+ and metabolites. Kinetic evidence for and against a metabolic pH-stat

1981 ◽  
Vol 59 (8) ◽  
pp. 1397-1404 ◽  
Author(s):  
Ceredwyn E. Smith ◽  
Alan W. Bown
Keyword(s):  
Enzyme Activity ◽  
Phosphoenolpyruvate Carboxylase ◽  
Malic Enzyme ◽  
Ph Regulation ◽  
Substrate Inhibition ◽  
Malic Enzyme Activity ◽  
Ph Stat ◽  
Phosphorylated Sugars ◽  
Cytosol Ph

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malic enzyme (EC 1.1.1.40) activities in soluble protein extracts of Avena coleoptiles were investigated as functions of pH. The presence of malic enzyme activity was confirmed by radiochemical assays which identified the product of the forward reaction and spectrophotometric assays which demonstrated that reduction or oxidation of NADP required all the substrates and cofactors necessary for the forward or reverse reactions. In neither assay could NADP or Mn2+ be replaced by NAD or Mg2+. Activity was independent of pH (6.0 to 7.7) at malate concentrations less than 0.1 mM. At higher concentrations the pH increase raised activity by at least 100%, and substrate inhibition by malate resulted in activity at 0.97 mM malate which was less than that at 0.5 mM malate. The increase in malic enzyme activity with rising pH can be attributed to a decrease in substrate inhibition and a decrease in Km,app (Mn2+). pH increases between 7.0 and 7.6 increased Vmax and phosphoenolpyruvate limited phosphoenolpyruvate carboxylase (PEPC) activity by over 200%, and decreased the Km,app (Mg2+). Inhibition of PEPC and malic enzyme by various carboxylic acids and phosphorylated sugars decreased as the assay pH rose. The results are discussed in light of contrasting suggestions concerning the role of these enzymes in cytosol pH regulation.

Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

1984 ◽  
Vol 42 ◽  
pp. 232-233
Author(s):  
S.M. Geyer ◽  
C.L. Mendenhall ◽  
J.T. Hung ◽  
E.L. Cardell ◽  
R.L. Drake ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Malic Enzyme ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Mature Male ◽  
Treatment Groups ◽  
Malic Enzyme Activity ◽  
Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

‘Malic Enzyme’ Activity in Blowfly Muscle

Nature ◽  
1956 ◽  
Vol 177 (4514) ◽  
pp. 842-843 ◽  
Author(s):  
S. E. LEWIS ◽  
G. M. PRICE
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Malic Enzyme Activity

An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse*

Development ◽  
1984 ◽  
Vol 80 (1) ◽  
pp. 251-288
Author(s):  
R. L. Gardner
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Cell Mass ◽  
Differential Staining ◽  
Wild Type ◽  
Primitive Endoderm ◽  
Extraembryonic Endoderm ◽  
Blue Tetrazolium ◽  
Malic Enzyme Activity ◽  
Parietal Endoderm

Conditions were found for staining whole mid-gestation capsular parietal endoderms and visceral yolk sacs for malic enzyme activity that gave excellent discrimination between wildtype (Mod-1+/Mod-1+) cells and mutant (Mod-ln/Mod-1n) cells that lack the cytoplasmic form of the enzyme. Reciprocal blastocyst injection experiments were undertaken in which single primitive endoderm cells of one genotype were transplanted into embryos of the other genotype. In addition, Mod-1+/Mod-1+ early inner cell mass (ICM) cells were injected into Mod-1n/Mod-1n blastocysts, either in groups of two or three singletons or as daughter cell pairs. A substantial proportion of the resulting conceptuses showed mosaic histochemical staining in the parietal endoderm, visceral yolk sac, or in both these membranes. Stained cells were invariably intimately intermixed with unstained cells in the mosaic parietal endoderms. In contrast, one or both of two distinct patterns of staining could be discerned in mosaic visceral yolk sacs. The first, a conspicuously ‘coherent’ pattern, was found to be due to endodermal chimaerism; the second, a more diffuse pattern, was attributable to chimaerism in the mesodermal layer of this membrane. The overall distribution of cells with donor staining characteristics resulting from primitive endoderm versus early ICM cell injections was consistent with findings in earlier experiments in which allozymes of glucosephosphate isomerase were used as markers. The conspicuous lack of phenotypically intermediate cells in predominantly stained areas of mosaic membranes suggested that the histochemical difference between Mod-1+/Mod-1+ and Mod-1n/Mod-ln genotypes was cell-autonomous. This conclusion was strengthened by the results of staining mixed in vitro cultures of parietal endoderm in which presence or absence of phagocytosed melanin granules was used as an independent means of distinguishing wild type from null cells. By substituting tetranitro blue tetrazolium for nitro blue tetrazolium in the incubation medium, satisfactory differential staining was obtained for both the extraembryonic endoderm and other tissues of earlier postimplantation wild type versus null embryos. Finally, absence of cytoplasmic malic enzyme activity does not appear to have a significant effect on the viability or behaviour of mutant cells.

scholarly journals Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

1998 ◽  
Vol 180 (11) ◽  
pp. 2875-2882 ◽  
Author(s):  
Eckhard Boles ◽  
Patricia de Jong-Gubbels ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Malic Enzyme ◽  
Fold Increase ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Oxidative Decarboxylation ◽  
Mrna Levels ◽  
Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

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