Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Abstract

Cell-free extracts from nodules of cowpea ( Vigna unguiculata L. (Walp.) cv Caloona:Rhizobium strain CB756) prepared in the presence of 15% (v/ v) glycerol showed high rates (30 to 60 nanomoles NAD reduced per minute per gram fresh weight nodule) of inosine monophosphate oxidoreductase (EC 1.2.1.14) activity. The enzyme was labile (half-life of activity less than 3 hours) but could be stabilized for up to 18 hours by inclusion of the substrates NAD and inosine monophosphate in the breaking media. Activity showed a broad pH optimum between 8.5 and 9.5, had an apparent K. (inosine monophosphate) of 4 and 12 micromolar at pH 7.5 and 9.0, respectively, and was largely (96%) associated with the plant ceil cytosol fraction of the nodule.Metabolism of 18-14Clinosine monophosphate and il-'4Ciglycine by the cell-free system showed two pathways for purine base production from inosine monophosphate, one via xanthosine monophosphate, xanthosine, and xanthine, the other via inosine and hypoxanthine. The proportion of inosine monophosphate utilized by inosine monophosphate oxidoreductase and the xanthine-based pathway was increased from 30% at 0.5 millimolar to 80% at 0.01 millimolar inosine monophosphate. The data are interpreted to indicate that in vivo inosine monophosphate oxidation rather than dephosphorylation is the predominant metabolic route leading to ureide synthesis and that inosine monophosphate provides the link between de novo purine nucleotide synthesis in the plastid and ureide production in the plant cell cytosol. doreductase (EC 1.2.1.37) in crude (4) or highly purified preparations (6) of nodules. Application of the inhibitor to intact plants results in xanthine accumulation in the nodules (4,8,10) consistent with metabolism through IMP oxidoreductase (EC 1.2.1.14) in vivo. In cell-free extracts, labeled hypoxanthine rather than xanthine accumulates from (1) Freshly harvested nodules from 4-to 6-week-old plants were washed with deionized H20 and crushed in a chilled mortar and pestle with I to 2 volumes of 50 mm Hepes-KOH buffer (pH 7.5), containing 25 mm KCI, 5 mM MgCl2, 1% (w/v) PVP, 15% (w/v) glycerol, 10 mm ME, 5 nms DTT, 2 mm GSH, and 0.25 M sucrose.

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using 15N2 and Allopurinol

“…1, B and C) These were l/Ao to l/2o those of xanthine, indicating that IMP is likely to be metabolized principally through IMP oxidoreductase rather than by an alternative route via inosine. This is consistent with the conclusions of earlier studies on cowpea nodules (21) based on kinetic properties ofthe purified component enzymes (6) and on the production of XMP in a cell-free system (21).…”

“…Merr.) nodules (4), together with specific purine nucleosidase (8) and enzymes of purine oxidation (IMP:oxidoreductase, xanthine oxidoreductase, and urate oxidase) (3,6,21). Although a number of studies using labeled N2 have confirmed that currently fixed nitrogen is utilized in ureide synthesis (12)(13)(14)(15)(16), none has reported the recovery of purine pathway intermediates as products of fixation.…”

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis

“…This is not surprising for the latter reaction because no inosine dehydrogenase has been identified in any species. However, XMP dephosphorylation has been postulated to occur when inosine monophosphate (IMP) is degraded to form ureides in nodules of ureide-exporting legumes (Shelp and Atkins, 1983) and for the generation of purine alkaloids (caffeine and theobromine) from IMP in coffee (Coffea arabica) and tea (Ito and Ashihara, 1999;Koshiishi et al, 2001;Keya et al, 2003), although direct evidence for XMP dephosphorylation in vivo is lacking. Nonetheless, all current reviews of plant nucleotide metabolism also include an XMP-to-xanthosine dephosphorylation reaction in their overview schemes (Stasolla et al, 2003;Zrenner et al, 2006;Ashihara et al, 2008).…”

Oligo-Carrageenans Stimulate Growth by Enhancing Photosynthesis, Basal Metabolism, and Cell Cycle in Tobacco Plants (var. Burley)

“…Inosine monophosphate dehydrogenase (IMPDH) activity was detected as described by Shelp and Atkins (1983) in 1 ml of reaction mixture containing 100 mM Tris-HCl (pH 7.2), 100 mM KCl, 2 mM inosine monophosphate, 1 mM NAD, 3 mM EDTA, 1 mM DTT, and 10 lg of protein extract. The increase in absorbance due to NADH synthesis was detected at 340 nm for 3 min.…”