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.
4.2.9 Synthesis of IMP via Formamidoimidazole Carboxamide Ribonucleotide
The last two steps to form IMP, the first complete purine nucleotide, are catalysed by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (abbreviated as ATIC). In the first part of this reaction the final carbon of the purine ring is provided by 10-formyl-THF to form 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR) (step 9, Figure 4.1, Reaction 9). FAICAR then undergoes dehydration and ring closure to generate IMP (step 10, Figure 4.1, Reaction 10).
4.2.10 Synthesis of AMP
AMP and GMP are synthesized from IMP. AMP is formed by replacing the carboxyl group at C6 with an amino group from aspartate. GTP is the donor for the energy-rich phosphate bond to form adenylosuccinate (SAMP) (step 11, Figure 4.1, Reaction 11). The reaction is catalysed by SAMP synthase (ASS, EC 6.3.4.4).
The cDNA encoding ASS (pur11) has been isolated and characterized from A. thaliana. This enzyme is a known target for herbicides and antibiotics (Fonné-Pfister et al. 1996). The structure of ASS has been investigated using recombinant ASS proteins from A. thaliana and Tritium aestivum expressed in E. coli. Comparison with the known structures from E. coli revealed that the overall fold is very similar to that of the E. coli protein. The longer N terminus in the plant sequences is at the same place as the longer C terminus of the E. coli sequence in the 3D structure. The GDP-binding sites have one additional hydrogen-bonding partner, which is a plausible explanation for the lower Km value (Prade et al. 2000).
The removal of fumarate to form AMP is catalysed by ASL (step 12, Figure 4.1, Reaction 12). ASL also catalyses step 8 of the purine biosynthesis de novo.
4.2.11 Synthesis of GMP
Synthesis of GMP is initiated by the oxidation of IMP followed by the insertion of an amino group that is provided by glutamine. Xanthosine-5′-monophosphate (XMP) formation
