Plant Nucleotide Metabolism. Hiroshi Ashihara

(3.5.4.10); (11) SAMP synthetase (6.3.4.4); (12) adenylosuccinate lyase (4.3.2.2), the same enzyme for step 8; (13) IMP dehydrogenase (1.1.1.205); (14) GMP synthetase (6.3.5.2).

      The nitrogen-fixing root nodules of Phaseolus vulgaris (common bean) also have three pur1 genes coding for PRAT (Coleto et al. 2016). One of the three pur1 genes which encodes PvPRAT3, is highly expressed in nodules and the enzymatic activity is correlated with nitrogen fixation activity. Some mutant studies suggest that PvPRAT3 is essential for the synthesis of ureides in nodules.

The formation of PRA from PRPP and glutamine occurs as two consecutive reactions that take place at separate active sites on PRAT.

      1 (i) glutamine + H2O → glutamate + NH3

      2 (ii) NH3 + PRPP → PRA + PPi

      Ammonia is released at the glutaminase domain and channelled to the PRPP binding site in the phosphoribosyltransferase domain (Zalkin and Dixon 1992). PRAT is structurally classified into two groups, namely, [4Fe-4S] cluster-dependent (type I) and independent-enzyme groups (type II). Plant PRAT displays the typical conserved protein structures of type I, containing a propeptide preceding the first cysteine and four conserved cysteine residues that are ligands to a [4Fe-4S] cluster and crucial for catalytic activity and feedback regulation (Ito et al. 1994).

      4.2.2 Synthesis of Glycineamide Ribonucleotide

      The second step of the de novo pathway is the ATP-dependent formation of glycineamide ribonucleotide (GAR) by the addition of glycine to PRA via an amide bond. This reaction is catalysed by glycineamide ribonucleotide synthetase (GARS, EC 6.3.4.13) (step 2, Figure 4.1, Reaction 2).

      The enzymatic properties of GARS are not well characterized. However, fluctuation in its activity during growth of cultured carrot cells has been investigated and the highest level was found in the cell division phase (Ashihara and Nygaard 1989).

      4.2.3 Synthesis of Formylglycineamide Ribonucleotide

      GAR is metabolised by the enzyme glycineamide ribonucleotide transformylase (GARFT, EC 2.1.2.2) using 10-formyltetrahydrofolate (10-Formyl-THF) to generate formylglycineamide ribonucleotide (FGAR) (step 3, Figure 4.1, Reaction 3).

Soybean nodule cDNA clones pur2 and pur3 encode enzymes that catalyse the second and the third steps of the de novo purine biosynthesis pathway, namely, GARS and GARFT, which were isolated by complementation of corresponding E. coli mutants. One class of the cDNA clone of GARS and the clones of three classes of GARFT cDNA were identified. These pur2 and pur3 genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves. Expression levels of the mRNAs of these two genes were not enhanced even when ammonia was provided to non-nodulated roots. (Schnorr et al. 1996).

      4.2.4 Synthesis of Formylglycinamidine Ribonucleotide

      FGAM synthesis from FGAR and glutamine is catalysed by formylglycinamide ribonucleotide amidotransferase (FGAMS, EC 6.3.5.3), in a reaction that requires ATP (step 4 in Figure 4.1, Reaction 4).

      The pur4 gene encodes the N-terminal portion of the FGAMS protein which contains targeting sequences of the FGAMS protein of A. thaliana suggesting that this enzyme is located in mitochondria as well as chloroplasts (Berthome et al. 2008). It has also been postulated that the FGAMS-catalysed step is crucial for plant reproduction, in particular male gametophyte development, but it probably also occurs in the sporophytic tissues sustaining pollen and embryo sac developments (Berthome et al. 2008).

      4.2.5 Synthesis of Aminoimidazole Ribonucleotide

      FGAM undergoes ring closure to form 5-aminoimidazole ribonucleotide (AIR) in a reaction that requires ATP (step 5, Figure 4.1, Reaction 5). This step is catalysed by aminoimidazole ribonucleotide synthase (AIRS, EC 6.3.3.1).

      The cDNA of pur5 encoding AIRS was identified and isolated from A. thaliana. The functional confirmation of the enzymatic activity was achieved by functional suppression of E. coli auxotrophs using expressed A. thaliana leaf cDNAs (Schnorr et al. 1994; Senecoff and Meagher 1993).

      4.2.6 Synthesis of Aminoimidazole Carboxylate Ribonucleotide

In contrast to animals, plants carboxylate AIR to 4-carboxy aminoimidazole ribonucleotide (CAIR) in a two-step reaction catalysed by aminoimidazole ribonucleotide carboxylase (AIRC, EC 4.1.1.21) (step 6, Figure 4.1, Reaction 6).

      This two-step reaction in plants is similar to that occurring in E. coli, where two separate enzymes, 5-(carboxyamino)imidazole ribonucleotide (N⁵-CAIR) synthase (PurK, EC 6.3.4.18), and N⁵-CAIR mutase (PurE, EC 5.4.99.18) are required to carry out the single reaction catalysed by AIRC (EC 4.1.1.21) in eukaryotes. In plants and yeast, PurK, and PurE proteins are fused and form an enzyme complex (Voet and Voet 2010). As in yeast, the moth bean (Vigna aconitifolia) AIRC has an N-terminal domain homologous to the bacterial purK gene product. This purK-like domain appears to facilitate the binding of HCO₃⁻ and is dispensable in the presence of high HCO₃⁻ concentrations (Chapman et al. 1994).

      In animals, activities of AIRC and the enzyme catalysing the next step, 4-(N-succinocarboxamide)-5-aminoimidazole synthetase (SAICARS), are associated with a single bifunctional polypeptide. However, these two enzymes are distinct proteins in plants (Chapman et al. 1994).

      4.2.7 Synthesis of Aminoimidazole Succinocarboxamide Ribonucleotide

      Conversion of CAIR to SAICAR is catalysed by SAICARS (EC 6.3.2.6). In this reaction, aspartate is added at the α-amino group of CAIR with the consumption of ATP (step 7 in Figure 4.1, Reaction 7).

The expression of the pur7 gene is strongest in flowers, followed by stem, root, and leaves and is almost absent in siliques and pollen. It has been reported that the expression of the gene is associated with actively dividing tissues such as meristems, and is responsive to growth hormones (Senecoff et al. 1996).

      4.2.8 Synthesis of Aminoimidazole Carboxamide Ribonucleotide

      Fumarate is released from SAICAR in the formation of aminoimidazole carboxamide ribonucleotide (AICAR) (step 8, Figure 4.1, Reaction 8). The reaction is catalysed by adenylosuccinate lyase (ASL, EC 4.3.2.2). This enzyme also catalyses the cleavage of adenylosuccinate leading to the production of AMP and fumarate (step 12). The encoding pur8/12 gene has been cloned from A. thaliana.