Plant Nucleotide Metabolism. Hiroshi Ashihara

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 is catalysed by inosine-5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) using NAD⁺ as the hydrogen acceptor (step 13, Figure 4.1, Reaction 13).

The native IMPDH has been investigated in seeds of pea (Pisum sativum) (Turner and King 1961), Catharanthus roseus cells and young leaves of tea (Camellia sinensis) (Nishimura and Ashihara 1993) and the soluble proteins of the plant cell fractionation of nitrogen-fixing nodules of cowpea (Vigna unguiculata). The most detailed studies have been with the cowpea enzyme which has an alkaline pH optimum (pH 8.8) and a high affinity for IMP and nicotinamide adenine dinucleotide (NAD). Intermediates of ureide metabolism do not affect the enzyme, while AMP, GMP, and NADH are inhibitors (Atkins et al. 1985). Similar to the cowpea nodule enzymes, the Km values of C. roseus and tea enzymes for IMP are low (∼20 μM) and inhibited by purine nucleotides, especially by GMP (Nishimura and Ashihara 1993). IMPDH is one of the key enzymes of caffeine biosynthesis (Keya et al. 2003). The full length cDNA (TIDH) encoding IMPDH was cloned from tea leaves and the expression of TIDH is highest in leaves where caffeine biosynthesis occurs than in other plant parts (Li et al. 2008). In addition to GMP and caffeine biosynthesis, the enzyme appears to contribute to the biosynthesis of ureides.

      The final step in the formation of GMP is catalysed by GMP synthase (GMPS, EC 6.3.5.2) (step 14, Figure 4.1, Reaction 14). Properties of this enzyme have not yet been reported.

4.3 Summary

      Reaction and enzymes of de novo purine nucleotide biosynthesis are described. There are 10 steps from PRPP to IMP. AMP and GMP are then produced by two steps from IMP.

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      16 Ito,