Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway

We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-P RT) from Arabidopsis (AtATP-PRT1 [accession no. AB025251] and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis. Th e primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast tra nsit peptide sequences. DNA-blot analyses indicated that the ATP-PRTs in Ar abidopsis are encoded by two separate genes with a closely similar gene str uctural organization. Both gene transcripts were detected throughout develo pment, and protein-blot analysis revealed predominant accumulation of the A tATP-PRT proteins in Arabidopsis leaves. The His auxotrophy of a his1 mutan t of Saccharomyces cerevisiae was suppressed by the transformation with AtA TP-PRT1 and AtATP-PRT2, cDNAs, indicating that both isoforms are functional ly active ATP-PRT enzymes. The K-m values for ATP and phosphoribosyl pyroph osphate of the recombinant AtATP-PRT proteins were comparable to those of t he native ATP-PRTs from higher plants and bacteria. It was demonstrated tha t the recombinant AtATP-PRTs were inhibited by L-His (50% inhibition of ini tial activity = 40-320 mu M), suggesting that His biosynthesis was regulate d in plants through feedback inhibition by L-His.