Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Appels, Michiel A.; Haaker, H.

doi:10.1104/pp.95.3.740

Cited by 60 publications

(17 citation statements)

References 21 publications

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“…A long-standing question concerning the excretion of amino acids by bacteroids of Rhizobium is the possible participation in shuttle mechanisms with the plant, such as the malate-aspartate shuttle (Kahn et af., 1985;McDermott et al, 1989 ;Appels & Haaker, 1991). While the data in this paper do not exclude such mechanisms it is apparent that amino acid excretion occurs in R. leguminosarum as a normal response to limitation of the TCA cycle, independently of the plant macrosymbiont.…”

Section: Discussionmentioning

confidence: 72%

“…Excretion of alanine but not glutamate has been detected for bacteroids from peas and soybeans (Appels & Haaker, 1991;Kouchi et al, 1991;Rosendahl et al, 1992). The pyruvate dehydrogenase complex has strong structural and functional similarities to the 2-oxoglutarate dehydrogenase complex (Miles & Guest, 1987;Guest & Russell, 1992), and its inhibition by a low redox potential, as has been suggested for the 2-oxoglutarate dehydrogenase complex, might lead to the accumulation of pyruvate or its transamination product alanine.…”

Section: Discussionmentioning

confidence: 99%

“…Consistent with these observations, the plant-derived peribacteroid membrane of Bradyrhizobium japonicum, which is relatively impermeable to sugars, has an active uptake system for dicarboxylic acids (Day & Udvardi, 1993). However, nutrient exchange may be more complex, since isolated bacteroids of R. leguminosarum and B. japonicum are known to excrete amino acids, particularly alanine and to a lesser extent aspartate (Appels & Haaker, 1991;Kouchi et al, 1991;Rosendahl et al, 1992). While on the one hand this has led to the proposal that a form of malate-aspartate shuttle may operate between the plant and bacteroid, the alternative explanation is that amino acids may be excreted to remove excess carbon and reducing equivalents (McDermott et al, 1989;Appels & Haaker, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

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Reading RG6 6AJ, UKMutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to s u d and suct), which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex ( s u d B ) . Mutation of either SUCC or SUCD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of s u d or suct), the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosamm was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (SUCD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and reductant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.

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Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

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“…Glutamate has been shown to be taken up and metabolized by bacteroids (9-11) but has not been found to be exported. However, alanine and aspartate have been shown to be exported from bacteroids of Rhizobium leguminosarum (12,13) and Bradyrhizobium japonicum (14), but the amounts measured were not considered sufficient to support the operation of a nutrient exchange system. The ammonia diffusion hypothesis remains as the model for nitrogen movement from bacteroid to plant as there is no evidence to directly support a nitrogen-containing compound other than ammonia for this role (15).…”

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confidence: 99%

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Waters

Hughes

Purcell

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

118

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Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobialeguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogencontaining compound excreted. Bacteroids incubated in the presence of 15 N 2 excreted alanine highly enriched in 15 N. The ammonium in these assays neither accumulated significantly nor was enriched in 15 N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.In 1956 (1), ammonium was shown to be the product of nitrogenase, the bacterial enzyme that reduces atmospheric dinitrogen. In free-living, nitrogen-fixing microorganisms, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase͞glutamate synthase pathway. Various transaminases utilize glutamate to generate all of the other amino acids, which are then used to synthesize proteins, nucleic acids and other nitrogen-containing molecules.In rhizobia-leguminous plant symbioses, the majority of the dinitrogen reduced by the microsymbionts, referred to as bacteroids, is transferred to the plant. Because the rhizobia are physically separated from the plant cytosol by a plant-derived symbiosome membrane, the ammonium must move across the bacteroid and symbiosome membranes. The ammonia diffusion hypothesis has been the prevalent model surmising the initial steps of leguminous nodule nitrogen transfer. In this model, the product of nitrogenase exists in two forms within the cell, ammonium [NH 4 ϩ ] and ammonia [NH 3 ], which are readily interconvertible. The ammonium ion cannot diffuse across membranes, but ammonia is believed to diffuse freely out of the bacteroid. Once outside the bacteroid but within the symbiosome space, the ammonia may either diffuse or be transported across the symbiosome membrane (2) where it is assimilated by the host plant into glutamate by the concerted action of glutamine synthetase and glutamate synthase. Glutamate serves as the central nitrogen metabolite in the plant nodule cells for the synthesis of the other amino acids, nucleic acids, and other nitrogen-containing compounds such as ureides that are the principal nitrogen compounds transported from the nodules to the plant shoots and leaves o...

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“…This catabolism could be direct or part of a more complicated pathway, such as the malate-aspartate shuttle found in mitochondria (1,32). The two soluble enzymes of this shuttle, AAT and malate dehydrogenase (1), are present in high levels in both the plant cytoplasm and bacteroid fractions in pea root nodules (49), as are their substrates. For such a shuttle to function in nodules, the carbon compounds involved must be transported across the symbiosome membrane and the bacteroid membrane.…”

Section: * Corresponding Authormentioning

confidence: 99%

Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti

Alfano

Kahn

1993

J Bacteriol

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Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, WttA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aaL4, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and hztA and transferred to the genome ofR. melioti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation.Bacteria of the genus Rhizobium fix nitrogen in a symbiotic association with leguminous plants. Carbon compounds produced by the plant are fed to the bacteria and metabolized to supply the energy and reductant needed for nitrogen fixation. Identifying which carbon compounds are transferred to the bacteria and how these compounds are metabolized is important in understanding the physiology of the symbiosis. Although sucrose is the major carbon source transported from the shoot to the nodule (44), most data suggest that it is not an important source of reductant for bacteroids (58) and that dicarboxylic acids are more likely to be their main carbon and energy sources (8,13,56). Succinate and malate are found in high concentrations in nodules (20,49,57) and bacteroids (15,29,45,51)

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Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Cited by 60 publications

References 21 publications

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti

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Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Cited by 60 publications

References 21 publications

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti

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Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids