A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg(-1), which corresponds to a turnover number of 1,055 min(-1). The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K(m) value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).
French bean (Phaseolus vulgaris) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.
Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the hydrolysis of allantoin to allantoic acid, a key reaction in the biosynthesis and degradation of ureides. This activity was determined in different tissues of French bean plants (Phaseolus vulgaris L.) which were grown under nitrogen-fixing conditions. Allantoinase activity was detected in all tissues analysed, but the highest levels of specific activity were found in developing fruits, from which allantoinase has been purified to electrophoretic homogeneity and further characterized. After diethylaminoethyl (DEAE)-Sephacel chromatography, two peaks showing allantoinase activity were obtained in the chromatographic profile and the corresponding proteins were independently purified. Total allantoinase activity was purified 200-fold, indicating the relevance of this enzymatic activity in French bean developing fruits, with allantoinase representing 0.5% of total soluble protein. Both proteins with allantoinase activity are monomeric with molecular masses of 45 and 42 kDa. The specific activities of the purified proteins were 560 and 295 units mg(-1), which correspond to turnover numbers of 25,200 and 12,100 min(-1), respectively. The two proteins have very similar biochemical properties showing Michaelis-Menten kinetics for allantoin with K(m) values of about 60 mM, with high optimal temperatures; are metalloenzymes; are inhibited by compounds reacting with sulphydryl groups; and are unaffected by reducing agents. All analysed tissues exhibited the two activities responsible for allantoin degradation, although one of them was the main form in leaves (the most photosynthetic tissue) and the other protein was the main form in roots (non-photosynthetic tissue). The allantoinase activity and distribution of both proteins have been analysed during fruit development. For both proteins, the allantoinase activity and distribution pattern were the same in plants growing either under nitrogen-fixing conditions or fertilized with nitrate.
In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined "in vivo" using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37 degrees C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.
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