Role of Pyruvate Carboxylase, Phosphoenolpyruvate Carboxykinase, and Malic Enzyme during Growth and Sporulation of Bacillus subtilis

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.

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Intracellular localization of pyruvate carboxylase, phosphoenolpyruvate carboxykinase and “malic enzyme” and the absence of glyoxylate cycle enzymes in the sea mussel (Mytilus edulis L.)

Comparative Biochemistry and Physiology Part B Comparative Biochemistry ◽

10.1016/0305-0491(73)90259-9 ◽

1973 ◽

Vol 44 (4) ◽

pp. 1057-1066 ◽

Cited By ~ 5

Author(s):

Albertus de Zwaan ◽

Willibrordus J.A. van Marrewijk

Keyword(s):

Mytilus Edulis ◽

Malic Enzyme ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Glyoxylate Cycle ◽

Intracellular Localization

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Role of pyruvate kinase, phosphoenolpyruvate carboxykinase, malic enzyme and lactate dehydrogenase in anaerobic energy metabolism ofTubifex spec

Journal of Comparative Physiology □ B ◽

10.1007/bf00689852 ◽

1979 ◽

Vol 130 (4) ◽

pp. 337-345 ◽

Cited By ~ 25

Author(s):

K. H. Hoffmann ◽

T. Mustafa ◽

J. B. J�rgensen

Keyword(s):

Lactate Dehydrogenase ◽

Energy Metabolism ◽

Pyruvate Kinase ◽

Malic Enzyme ◽

Phosphoenolpyruvate Carboxykinase ◽

Anaerobic Energy

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Identification of In Vivo Enzyme Activities in the Cometabolism of Glucose and Acetate by Saccharomyces cerevisiae by Using 13C-Labeled Substrates

Eukaryotic Cell ◽

10.1128/ec.2.3.599-608.2003 ◽

2003 ◽

Vol 2 (3) ◽

pp. 599-608 ◽

Cited By ~ 44

Author(s):

Margarida Moreira dos Santos ◽

Andreas Karoly Gombert ◽

Bjarke Christensen ◽

Lisbeth Olsson ◽

Jens Nielsen

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activities ◽

Malic Enzyme ◽

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Central Carbon Metabolism ◽

Isopropylmalate Synthase ◽

Very High

ABSTRACT A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with 13C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO2 via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that α-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

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A putative pathway of glyconeogenesis in skeletal muscle

Bioscience Reports ◽

10.1007/bf01117013 ◽

1981 ◽

Vol 1 (2) ◽

pp. 157-165 ◽

Cited By ~ 6

Author(s):

Bhanu R. Odedra ◽

T. Norman Palmer

Keyword(s):

Skeletal Muscle ◽

Malic Enzyme ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

De Novo ◽

Glycogen Biosynthesis ◽

Glycogen Sparing ◽

Specific Inhibitors

Evidence is presented in support of a pathway in skeletal muscle of glyconeogenesis (glycogen biosynthesis de novo) from L-glutamate and related amino acids involving the enzyme phosphoenolpyruvate carboxykinase (PEP CK). In the rat hemidiaphragm in vitro, not only did L-[U-14C]glutamate exert a glycogen-sparing action, but14C-label was incorporated into glycogen. The incorporation is thought not to be simply via label randomization and was decreased by factors that increased glycolysis or pyruvate oxidation. 3-Mercaptopicolinate and amino-oxyacetate, specific inhibitors of PEP CK and aminotransferase-type enzymes, respectively, decreased14C-incorporation from L-[U-14C]glutamate into glycogen. No quantitative determination of apparent glyconeogenic flux was made, and it remains to be established whether glyconeogenesis via PEP CK and/or via PEP CK coupled with 'malic' enzyme (or pyruvate carboxylase) is functionally important in skeletal muscle.

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Pyruvate carboxylation in the rat heart. Role of biotin-dependent enzymes

Biochemical Journal ◽

10.1042/bj2570913 ◽

1989 ◽

Vol 257 (3) ◽

pp. 913-916 ◽

Cited By ~ 22

Author(s):

K E Sundqvist ◽

J K Hiltunen ◽

I E Hassinen

Keyword(s):

Heart Muscle ◽

Malic Enzyme ◽

Rat Heart ◽

Pyruvate Carboxylase ◽

Mass Action ◽

Kinetic Properties ◽

Biotin Deficiency ◽

Perfused Rat Heart ◽

Pyruvate Carboxylation

Pyruvate carboxylation in the isolated perfused rat heart was studied under steady-state conditions. A biotin deficiency resulting in a 90% decrease in myocardial pyruvate carboxylase left the pyruvate carboxylation rate unchanged. Pyruvate carboxylation in heart muscle must therefore take place by means of an enzyme which does not contain biotin. The kinetic properties and mass-action ratio of the NADP-linked malic enzyme in heart muscle can be taken as circumstantial evidence in favour of the role of malic enzyme in pyruvate carboxylation in myocardium.

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Carbon dioxide fixation in trypanosomatids

Parasitology ◽

10.1017/s003118200005318x ◽

1975 ◽

Vol 71 (1) ◽

pp. 93-107 ◽

Cited By ~ 46

Author(s):

R. A. Klein ◽

D. J. Linstead ◽

M. V. Wheeler

Keyword(s):

Carbon Dioxide ◽

Subcellular Localization ◽

Trypanosoma Brucei ◽

Malic Enzyme ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Trypanosoma Brucei Brucei ◽

Short And Long Term ◽

Fixation Of Carbon Dioxide

Fixation of carbon dioxide has been demonstrated for extracts from Crithidia fasciculata, Trypanosoma mega and Trypanosoma brucei brucei bloodstream and culture forms. The enzymes involved in this fixation were found to be ADP-stimulated phosphoenolpyruvate carboxykinase (E.C. 4. 1. 1. 32), ‘malic’ enzyme (E.C. 1. 1. 138–40) and pyruvate carboxylase (E. 0. 6. 4. 1. 1). The subcellular localization of these enzymes has been investigated in all three organisms. Products of short and long term fixation experiments were separated and identified.The importance of carboxylation reactions is discussed in relation to the maintenance of oxidized and reduced coenzyme levels.

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Renal enzymes during experimental diabetes mellitus in the rat. Role of insulin, carbohydrate metabolism, and ketoacidosis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y84-010 ◽

1984 ◽

Vol 62 (1) ◽

pp. 70-75 ◽

Cited By ~ 26

Author(s):

Guy Lemieux ◽

Manuel Rengel Aranda ◽

Pierrette Fournel ◽

Christiane Lemieux

Keyword(s):

Lactate Dehydrogenase ◽

Glutamine Synthetase ◽

Glutamate Dehydrogenase ◽

Malate Dehydrogenase ◽

Ammonium Chloride ◽

Malic Enzyme ◽

Phosphoenolpyruvate Carboxykinase ◽

Diabetic Rats ◽

Additional Role

The activities of various ammoniagcnic, gluconeogenic, and glycolytic enzymes were measured in the renal cortex and also in the liver of rats made diabetic with streptozotocin. Five groups of animals were studied: normal, normoglycemic diabetic (insulin therapy), hyperglycemic, ketoacidotic, and ammonium chloride treated rats. Glutaminase I, glutamate dehydrogenase, glutamine synthetase, phosphoenolpyruvate carboxykinase (PEPCK), hexokinase, phosphofructokinase, fructose-1,6-diphos-phatase, malate dehydrogenase, malic enzyme, and lactate dehydrogenase were measured. Renal glutaminase I activity rose during ketoacidosis and ammonium chloride acidosis. Glutamate dehydrogenase in the kidney rose only in ammonium chloride treated animals. Glutamine synthetase showed no particular variation. PEPCK rose in diabetic hyperglycemic animals and more so during ketoacidosis and ammonium chloride acidosis. It also rose in the liver of the diabetic animals. Hexokinase activity in the kidney rose in diabetic insulin-treated normoglycemic rats and also during ketoacidosis. The same pattern was observed in the liver of these diabetic rats. Renal and hepatic phosphofructokinase activities were elevated in all groups of experimental animals. Fructose-1,6-diphosphatase and malate dehydrogenase did not vary significantly in the kidney and the liver. Malic enzyme was lower in the kidney and liver of the hyperglycemic diabetic animals and also in the liver of the ketoacidotic rats. Lactate dehydrogenase fell slightly in the liver of diabetic hyperglycemic and NH4Cl acidotic animals. The present study indicates that glutaminase I is associated with the first step of increased renal ammoniagenesis during ketoacidosis. PEPCK activity is influenced both by hyperglycemia and ketoacidosis, acidosis playing an additional role. Insulin appears to prevent renal gluconeogenesis and to favour glycolysis. The latter would seem to remain operative in hyperglycemic and ketoacidotic diabetic animals.

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