Molecular Cloning and Characterization of ATP-PhosphoribosylTransferase from Arabidopsis a Key Enzyme in the HistidineBiosynthetic Pathway

摘要

We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-PRT) from Arabidopsis (AtATP-PRT1 [accession no. ] and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis. The primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast transit peptide sequences. DNA-blot analyses indicated that the ATP-PRTs in Arabidopsis are encoded by two separate genes with a closely similar gene structural organization. Both gene transcripts were detected throughout development, and protein-blot analysis revealed predominant accumulation of the AtATP-PRT proteins in Arabidopsis leaves. The His auxotrophy of a his1 mutant of Saccharomyces cerevisiae was suppressed by the transformation with AtATP-PRT1 and AtATP-PRT2 cDNAs, indicating that both isoforms are functionally active ATP-PRT enzymes. The Kmvalues for ATP and phosphoribosyl pyrophosphate of the recombinantAtATP-PRT proteins were comparable to those of the native ATP-PRTs fromhigher plants and bacteria. It was demonstrated that the recombinantAtATP-PRTs were inhibited by l-His (50% inhibition ofinitial activity = 40–320 μm), suggesting that Hisbiosynthesis was regulated in plants through feedback inhibition byl-His.