thaliana, as well as in the ureide-forming tropical legumes. However, there are many difficulties in predicting the actual localization of the enzymes. For example, a number of proteins that have been identified are directed to plastids and mitochondria by the same targeting sequence (Chow et al. 1997; Menand et al. 1998).
Information in the literature on purine metabolism, especially de novo biosynthesis in plants, is fragmentary. However, it is possible to follow progress using databases linked to the TAIR gene locus numbers of A. thaliana as well as the EC numbers (the International Union of Biochemistry and Molecular Biology) shown in Table 4.1. Useful databases for nucleotide metabolism are KEGG (Kyoto Encyclopedia of Genes and Genomes), BRENDA (The Comprehensive Enzyme Information System), and MetaCyc (BioCyc Database Collection).
4.2.1 Synthesis of Phosphoribosylamine
Purine biosynthesis de novo starts with the formation of phosphoribosylamine (PRA) from PRPP and glutamine (step 1, Figure 4.1, Reaction 1).
This reaction is catalysed by 5-phosphoribosyl-1-pyrophosphate amidotransferase (PRAT, EC 2.4.2.14). PRAT has been purified from a number of sources, including E. coli, and Bacillus subtilus, avian liver, and human placenta. In plants, a highly purified native enzyme has been obtained from root nodules of soybean (Glycine max) (Reynolds et al. 1984). In tropical legumes, ammonia, a direct product of symbiotic nitrogen fixation in roots, is converted to ureides (allantoin and allantoic acid) which are translocated to the shoot where they serve as a nitrogen source. Ureides are generated through the oxidation of purines formed by the de novo purine biosynthetic pathway. PRAT catalyses the first committed step of ureide biosynthesis in soybean. Nodule PRAT has a pH optimum of 8.0, and Km values of 18 mM for glutamine and 0.4 mM for PRPP. Ammonia can replace glutamine as a substrate (Km ∼14 mM) and the Vmax with ammonia is double that obtained when glutamine is the substrate. Regulatory properties of the soybean root nodule enzyme are similar to those reported for the human placental enzyme (Holmes et al. 1973). Both are subject to regulation by end products of the purine biosynthetic pathway, IMP, xanthosine-5′-monophosphate (XMP), guanosine-5′-monophosphate (GMP), and adenosine-5′-monophosphate (AMP). Therefore, the first step of purine biosynthesis de novo is feedback controlled by the end products in both plants and animals.
In plants, there are three isoforms of PRAT (AtPRAT1–3) which are coded in a family of three isoforms of pur1. In A. thaliana, AtPRAT2 is the major isoform (Hung et al. 2004). One of the pur1 genes which encodes AtPRAT1 protein is expressed in roots and flowers. The AtPRAT2-encoding gene is expressed in leaves as well as roots and flowers (Ito et al. 1994), while the gene that encodes AtPRAT3 is expressed at very low levels in silique, cauline leaves, and roots (Hung et al. 2004).
Figure 4.1 Purine ribonucleotide biosynthesis. Abbreviations of metabolites are as follows: PRPP, 5-phosphoribosyl-1-pyrophosphate; PRA, 5-phosphoribosyl amine; GAR, glycineamide ribonucleotide; FGAR, formylglycineamide ribonucleotide; FGAM, formylglycinamidine ribonucleotide; AIR, 5-aminoimidazole ribonucleotide; CAIR, 5-aminoimidazole 4-carboxylate ribonucleotide; SAICAR, 5-aminoimidazole-4-N-succinocarboxyamide ribonucleotide; AICAR, 5-aminoimidazole-4-carboxyamide ribonucleotide; FAICAR, 5-formamidoimidazole-4-carboxyamide ribonucleotide; SAMP, adenylosuccinate; XMP, xanthosine-5′-monophosphate; IMP, inosine-5′-monophosphate; GMP, guanosine-5′-monophosphate; AMP, adenosine-5′-monophosphate; Asp, aspartate; Gln, glutamine; Gly, glycine; 10-THF, 10-formyltetrahydrofolate. The participated enzymes are (1) PRPP amidotransferase (2.4.2.14); (2) GAR synthetase (6.3.4.13); (3) GAR formyl transferase (2.1.2.2); (4) FGAM synthetase (6.3.5.3); (5) AIR synthetase (6.3.3.1); (6) AIR carboxylase (4.1.1.21); (7) SAICAR synthetase (6.3.2.6); (8) adenylosuccinate lyase (4.3.2.2); (9) AICAR formyl transferase (2.1.2.3); (10) IMP cyclohydrolase (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
