Chimeric Structure of the NAD(P)+- and NADP+-dependent Malic Enzymes ofRhizobium (Sinorhizobium) meliloti

Mitsch, Michael J.; Voegele, Ralf T.; Cowie, Alison; Østerås, Magne; Finan, Turlough M.

doi:10.1074/jbc.273.15.9330

Cited by 32 publications

(56 citation statements)

References 51 publications

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“…The gene b2463 (EMBL accession no. AE000333) was suggested previously to encode an NADP-dependent malic enzyme (21), but no experimental data were provided. Deletion of b2463 from the E. coli genome abolishes NADP-dependent malic enzyme activity (results not shown), thereby justifying the EC 1.1.1.40 designation for the gene product of b2463.…”

Section: Hydrolysis Of Ortho-nitrophenyl-␤-d-galactopyranoside (Onpg)mentioning

confidence: 99%

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

2000

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Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinoneoxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the ␤-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.In Escherichia coli, several enzymes or pathways are able to convert malate to oxaloacetate. The NAD-dependent (cytoplasmic) malate dehydrogenase (MDH; EC 1.1.1.37) has always been considered to be the principal malate-oxidizing enzyme in the citric acid cycle (tricarboxylic acid [TCA] cycle) of this organism (9). Recently, a malate:quinone-oxidoreductase (MQO; EC 1.1.99.16) which is an essential enzyme in the TCA cycle of Corynebacterium glutamicum (reference 23 and accompanying paper) was described. MQO is a flavin adenine dinucleotide (FAD)-and lipid-dependent peripheral membrane protein catalyzing the oxidation of L-malate to oxaloacetate. The electrons are donated to the electron transfer chain at the level of quinones. This reaction is essentially irreversible (17). In C. glutamicum, MQO and the cytoplasmic NAD-dependent malate dehydrogenase, which is capable of reversible oxidation of malate to oxaloacetate, are active at the sam...

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Section: Hydrolysis Of Ortho-nitrophenyl-␤-d-galactopyranoside (Onpg)mentioning

confidence: 99%

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

2000

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“…When nodule C 4 -dicarboxylic acids have been measured, L-malate has proved the most abundant, suggesting that its oxidation is a key step in carbon flux in the bacteroid (30)(31). Consistent with this, NAD ϩ malic enzyme (diphosphopyridine nucleotide-dependent malic enzyme; Dme) in Sinorhizobium meliloti is essential for N 2 fixation in alfalfa nodules, but NADP ϩ malic enzyme (triphosphopyridine nucleotide-dependent malic enzyme; Tme) is not (3,5,(17)(18). This indicates that L-malate imported into the bacteroid is either oxidized by malate dehydrogenase to oxaloacetate or oxidatively decarboxylated to pyruvate by Dme.…”

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confidence: 91%

Pyruvate Is Synthesized by Two Pathways in Pea Bacteroids with Different Efficiencies for Nitrogen Fixation

et al. 2010

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Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD ؉ malic enzyme (Dme) but not NADP ؉ malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N 2 fixation, but the PckA/PykA pathway supports N 2 fixation at only 60% of that for Dme. Double mutants of dme and pckA/pykA did not fix N 2 . Furthermore, dme pykA double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N 2 fixation. When PckA was expressed from a constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.The largest input of available nitrogen in the biosphere comes from the biological reduction of atmospheric N 2 to ammonium (20). Most of this comes from legume-Rhizobium symbioses, which arise from infection of host plants and result in root nodules (21). Signaling between the plant and bacterium is initiated by plant-released flavonoids and related compounds, which elicit the synthesis of lipochitooligosaccharide Nod factors by rhizobia (21). Bacteria are trapped by curling root hairs that they enter via infection threads. These grow into a zone of newly induced meristematic cells in the cortex that form the origin of the nodule. Bacteria are released from infection threads by endocytosis and surrounded by a plantderived symbiosome membrane. Plants provide differentiated bacteria (bacteroids) with dicarboxylic acids which energize N 2 reduction to ammonium for secretion back to the plant (24, 37). A simple exchange of dicarboxylates and ammonium is the classical model of nutrient exchange in nodules, but amino acid transport by pea bacteroids has also been shown to be essential (13). This is because bacteroids reduce the synthesis of branched-chain amino acids and become symbiotic auxotrophs that depend on their provision by the plant (25). Thus, when the two broad-specificity amino acid ABC trans...

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“…The combined deletion of the two genes leads to a severe growth defect only on C 4 -dicarboxylates, thus indicating that malic enzyme activity is necessary under such growth conditions in E. coli (van der Rest et al, 2000). Rhizobium meliloti, a nitrogen-fixing Gram-negative symbiont of alfalfa, also synthesizes two malic enzymes named DME and TME, which are NAD-and NADP-dependent, respectively (Driscoll & Finan, 1997;Mitsch et al, 1998). The former has been shown to be involved in symbiotic nitrogen fixation as a result of its role for growth in the nodule, where C 4 -dicarboxylates are the major carbon and energy source provided by the host (Driscoll & Finan, 1993;Dunn, 1998), while the latter has been proposed to function as a generator of NADPH necessary for biosynthesis reactions (Driscoll & Finan, 1996).…”

Section: Introductionmentioning

confidence: 99%

The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate

et al. 2003

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A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k cat/K m 102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

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Chimeric Structure of the NAD(P)+- and NADP+-dependent Malic Enzymes ofRhizobium (Sinorhizobium) meliloti

Cited by 32 publications

References 51 publications

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

Pyruvate Is Synthesized by Two Pathways in Pea Bacteroids with Different Efficiencies for Nitrogen Fixation

The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate

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