MOLECULAR CHARACTERIZATION OF ARABIDOPSIS-THALIANA CDNAS ENCODING 3 PURINE BIOSYNTHETIC-ENZYMES

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and am inoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 16-step de novo purine biosynthetic pathway. Fro m a cDNA library of Arabidopsis thaliana, cDNAs encoding the above thr ee enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transform ylase or AIR synthetase. Comparisons of the three Arabidopsis purine b iosynthetic enzymes with corresponding enzymes/polypeptide-fragments f rom procaryotic and eucaryotic sources indicate a high deg ree of cons erved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR sy nthetase and GAR transformylase. Sequence analysis, as well as Norther n blot analysis indicate that Arabidopsis has single and monofunctiona l enzymes. In this respect the organization of these three plant purin e biosynthesis genes is fundamentally different from the multifunction al purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in proca ryotes.