Isolation and Characterization of a Histidine Biosynthetic Gene
in Arabidopsis Encoding a Polypeptide with Two Separate Domains for
Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP
Cyclohydrolase |
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Authors: | Ko Fujimori and Daisaku Ohta |
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Affiliation: | Takarazuka Research Institute, Novartis Pharma K.K., 10-66 Miyuki-cho, Takarazuka 665-8666, Japan (K.F.);Research Institute for Biological Sciences, Okayama, 7549-1 Yoshikawa, Kayo-cho, Okayama 716-1241, Japan (D.O.) |
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Abstract: | Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein. |
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