Products of Biological Nitrogen Fixation in Higher Plants: Synthesis, Transport, and Metabolism

Schubert, Karel R.

doi:10.1146/annurev.pp.37.060186.002543

Cited by 355 publications

(63 citation statements)

References 112 publications

(66 reference statements)

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“…The conversion of inorganic nitrogen into organic forms is an energetically expensive process, however. It has been estimated that 68 ATPs are required for the synthesis of allantoin if the cost of N 2 fixation is included (2). Thus, one would expect mechanisms for the efficient utilization of fixed nitrogen to have arisen.…”

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confidence: 99%

Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme

Sarma

Serfózó

Kahn

et al. 1999

Journal of Biological Chemistry

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We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules ϳ100-fold to apparent homogeneity with a final specific activity of 10 mol/ min/mg. The enzyme exhibited a native molecular mass of ϳ68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 ؎ 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K m for 5-hydroxyisourate was determined to be 15 M. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.Nitrogen is a key component of plant metabolism, and its availability often limits the growth of important crop plants. Leguminous plants are able to acquire their nitrogen through association with bacterial symbionts in the root nodules, which fix atmospheric nitrogen to form NH 4 ϩ through the action of nitrogenase. The fixed nitrogen is then transported from the bacteria into the host cell cytoplasm, where it is assimilated into organic form and used for the synthesis of nucleic acids, amino acids, and secondary products.The ureides are the major form of nitrogen transport molecules in tropical legumes such as soybean. In nodulated soybean ureides constitute 70 -80% of the organic nitrogen in the xylem sap (1). The ureides are efficient nitrogen transport species; the ratio of C to N in allantoin and allantoate is 1:1, so minimal carbon is diverted from other metabolic functions in support of nitrogen transport. The conversion of inorganic nitrogen into organic forms is an energetically expensive process, however. It has been estimated that 68 ATPs are required for the synthesis of allantoin if the cost of N 2 fixation is included (2). Thus, one would expect mechanisms for the efficient utilization of fixed nitrogen to have arisen.Purine oxidation is the major route for ureide biogenesis, and the so-called ureide pathway is constituted by the enzymes that carry out the conversion of IMP to allantoin and allantoate. It is commonly considered that the role of urate oxidase in this pathway is the conversion of urate to allantoin (3). However, it has recently been demonstrated that allantoin is not the true product of the urate oxidase reaction (4, 5). Urate oxidase catalyzes the conversion of urate to 5-hydroxyisourate, which decomposes cleanly to allantoin under most in vitro conditions.The half-life of HIU 1 at neutral pH is on the order of 30 min in vitro (6). Because the flux through the ureide pathway is critical for nitrogen fi...

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confidence: 99%

Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme

Sarma

Serfózó

Kahn

et al. 1999

Journal of Biological Chemistry

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“…and reducing equivalents between the cytoplasm and organelles via a malate-aspartate shuttle,2) and in assimilation of ammonia produced by the endosymbiotic rhizobia. 3 ) Plants contain several isoenzymes of AAT. Two forms of AAT have been identified in alfalfa 4 ) and Eleusine coracana 5 ) and purified partially or homogeneously.…”

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Purification and Characterization of Aspartate Aminotransferase Isoenzymes from Rice Bran

Yagi

Sako

Moriuti

et al. 1993

Bioscience, Biotechnology, and Biochemistry

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“…Fixation of N2 is an energyintensive process, requiring a total of [25][26][27][28][29][30] ATP per N atom fixed. Of this total, [12][13][14] per N are required within the bacteroid to reduce N2.…”

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Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

Kohl

Schubert

Carter

et al. 1988

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

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N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides).The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vm. with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but <2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. N2 fixation in legumes is a symbiotic process in which bacteria of the genus Bradyrhizobium or Rhizobium infect root cells and form specialized organs (nodules) within which N2 is reduced to NH'. Fixation of N2 is an energyintensive process, requiring a total of 25-30 ATP per N atom fixed. Of this total, 12-14 ATP per N are required within the bacteroid to reduce N2. As much as 10-30% of the total photosynthetic capacity of the plant is used to support this process (1). The energy-yielding metabolite(s) supplied by the host to the bacteroid, the symbiotic form of the bacterium, is not known. However, one attractive suggestion is that bacteroids import and oxidize a nitrogenous compound, such as glutamate (2). The nitrogen fixed in the bacteroid is exported as ammonia(um) to the infected host cell, where it is packaged for export to the rest of the plant. Legumes of temperate origin (e.g., peas) export nitrogen as amides (principally asparagine), whereas legumes of tropical origin (e.g., soybeans) export the ureides, allantoin, and allantoic acid. Ureide biogenesis proceeds by way of synthesis of purine ribonucleotides (3). This pathway is the same as that found in microorganisms, fungi, and animals. The purines so formed are then oxidatively degraded to ureides (3). The estimated peak rate of de novo purine biosynthesis necessary t...

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Products of Biological Nitrogen Fixation in Higher Plants: Synthesis, Transport, and Metabolism

Cited by 355 publications

References 112 publications

Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme

Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme

Purification and Characterization of Aspartate Aminotransferase Isoenzymes from Rice Bran

Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

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