Stepwise versus Concerted Oxidative Decarboxylation Catalyzed by Malic Enzyme: A Reinvestigation

Biochemistry ◽  
1994 ◽  
Vol 33 (8) ◽  
pp. 2096-2103 ◽  
Author(s):  
William E. Karsten ◽  
Paul F. Cook
Keyword(s):  
Malic Enzyme ◽  
Oxidative Decarboxylation

Alternative Substrates for Malic Enzyme:  Oxidative Decarboxylation ofl-Aspartate†

Biochemistry ◽  
2002 ◽  
Vol 41 (40) ◽  
pp. 12200-12203 ◽  
Author(s):  
Dali Liu ◽  
Chi-Ching Hwang ◽  
Paul F. Cook
Keyword(s):  
Malic Enzyme ◽  
Oxidative Decarboxylation ◽  
Alternative Substrates

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.

scholarly journals Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

2020 ◽  
Author(s):  
Diego Rivera Gelsinger ◽  
Rahul Reddy ◽  
Kathleen Whittington ◽  
Sara Debic ◽  
Jocelyne DiRuggiero
Keyword(s):  
Oxidative Stress ◽  
Malic Enzyme ◽  
Tricarboxylic Acid Cycle ◽  
Redox Homeostasis ◽  
Fine Tuning ◽  
Oxidative Decarboxylation ◽  
Seed Region ◽  
Stem Loop ◽  
Loop Region ◽  
Rna Interaction

ABSTRACTHaloarchaea are highly resistant to oxidative stress, however, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volcanii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative “seed” region within the predicted stem loop of SHOxi. Malic enzyme is an enzyme of the tricarboxylic acid cycle that catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

scholarly journals Characterization of an archaeal malic enzyme from the hyperthermophilic archaeonThermococcus kodakaraensisKOD1

Archaea ◽  
2005 ◽  
Vol 1 (5) ◽  
pp. 293-301 ◽  
Author(s):  
Wakao Fukuda ◽  
Yulia Sari Ismail ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Hyperthermophilic Archaea ◽  
Oxidative Decarboxylation ◽  
Limited Information ◽  
Gene Encoding ◽  
Malic Enzyme Activity ◽  
Reductive Carboxylation ◽  
Reaction Direction

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeonT hermococcus kodakaraensisKOD1 (Tk-Mae). The amino acid sequence ofTk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure ofTk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) ofTk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encodingTk-Mae and intracellular malic enzyme activity inT. kodakaraensiswere constitutively weak, regardless of the growth substrates. Possible roles ofTk-Mae are discussed based on these results and the metabolic pathways ofT. kodakaraensisdeduced from the genome sequence.

scholarly journals Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties

1987 ◽  
Vol 245 (2) ◽  
pp. 407-414 ◽  
Author(s):  
H J Lee ◽  
G G Chang
Keyword(s):  
Synergistic Effect ◽  
Nicotinamide Adenine Dinucleotide ◽  
Malic Enzyme ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nucleotide Binding ◽  
Phosphate Group ◽  
Nicotinamide Adenine ◽  
Oxidative Decarboxylation ◽  
Wide Range ◽  
Pigeon Liver

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5′-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3′-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2′-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2′-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.

Kinetic mechanism in the direction of oxidative decarboxylation for NAD-malic enzyme from Ascaris suum

Biochemistry ◽  
1984 ◽  
Vol 23 (23) ◽  
pp. 5446-5453 ◽  
Author(s):  
Sang Hoon Park ◽  
Dennis M. Kiick ◽  
Ben G. Harris ◽  
Paul F. Cook
Keyword(s):  
Malic Enzyme ◽  
Ascaris Suum ◽  
Kinetic Mechanism ◽  
Oxidative Decarboxylation

scholarly journals The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

2009 ◽  
Vol 74 (8-9) ◽  
pp. 893-906 ◽  
Author(s):  
Veronika Doubnerová ◽  
Lucie Potůcková ◽  
Karel Müller ◽  
Helena Ryslavá
Keyword(s):  
Malic Enzyme ◽  
Citric Acid Cycle ◽  
Catalytic Mechanism ◽  
Enzyme Reaction ◽  
Defense Responses ◽  
Oxidative Decarboxylation ◽  
Plant Defense Responses ◽  
Tobacco Leaves ◽  
Nicotiana Tabacum L ◽  
Rate Method

The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME), which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (?-ketoglutarate, succinate, fumarate), metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate), compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA) and some amino acids (glutamate, glutamine, aspartate) did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP) were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+), required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

scholarly journals Anaplerotic Role for Cytosolic Malic Enzyme in EngineeredSaccharomyces cerevisiaeStrains

2010 ◽  
Vol 77 (3) ◽  
pp. 732-738 ◽  
Author(s):  
Rintze M. Zelle ◽  
Jacob C. Harrison ◽  
Jack T. Pronk ◽  
Antonius J. A. van Maris
Keyword(s):  
Metabolic Engineering ◽  
Adaptive Evolution ◽  
Malic Enzyme ◽  
Pyruvate Carboxylase ◽  
Single Point ◽  
Pyruvate Decarboxylase ◽  
High Yield ◽  
Single Point Mutation ◽  
Oxidative Decarboxylation ◽  
Favorable Conditions

ABSTRACTMalic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2. TheSaccharomyces cerevisiae MAE1gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylase-negative (Pyc−)S. cerevisiaestrains to grow on glucose suggested that Mae1p cannot act as a pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme inS. cerevisiae. TheEscherichia coliNADH-dependentsfcAmalic enzyme was expressed in a Pyc−S. cerevisiaebackground. WhenPDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO2atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h−1). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference ofE. colimalic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in apyc1,2Δ pdc2Δ strain grown under a CO2atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering ofS. cerevisiaefor anaerobic, high-yield C4-dicarboxylic acid production.

REGULATION OF THE "MALIC" ENZYME IN NEUROSPORA CRASSA

1967 ◽  
Vol 13 (9) ◽  
pp. 1211-1221 ◽  
Author(s):  
M. W. Zink
Keyword(s):  
Cell Growth ◽  
Neurospora Crassa ◽  
Growth Phase ◽  
Pyruvic Acid ◽  
Malic Acid ◽  
Malic Enzyme ◽  
Enzyme Preparation ◽  
Growth Cycle ◽  
Oxidative Decarboxylation ◽  
Enzyme Repression

"Malic" enzyme has been isolated from Neurospora crassa which can bring about the reversible carboxylation of pyruvic acid. The enzyme is specific to L-malate and NADP and is activated by Mn++ and Mg++. The partially purified enzyme does not decarboxylate oxaloacetate but is competitively inhibited by it. This enzyme is synthesized only during the early stages of the growth cycle and is repressed by acetate. In addition, the oxidative decarboxylation of malic acid is competitively inhibited by aspartic acid; the degree of inhibition depends upon the cell growth phase from which the enzyme is extracted. "Malic" enzyme isolated from a 12-h culture is not significantly inhibited by aspartate. However, this inhibition increases to 90% if an enzyme preparation from a 24-h culture is used. The significance of enzyme repression by acetate and the inhibition of the activity by aspartate are discussed.

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