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

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.

Figure 1

His biosynthetic pathway. The pathway starts with PRPP and ATP as the initial substrates. Box, The reactions catalyzed by the HIS4 of S. cerevisiae and the hisIE of E. coli.

Figure 2

Complementation of the His auxotrophy of E. coli strain UTH903 (hisI) by the Arabidopsis At-IE cDNA. E. coli UTH903 (hisI) was transformed with either an empty pBluescript SK(−) plasmid (pBS SK[−]) or a pBluescript SK(−) carrying a 1.1-kb At-IE cDNA (pAt-IE), streaked onto an M9-Glc minimal agar plate in the presence (M9+His) or absence (M9) of 1 mm l-His, and incubated overnight at 37°C.

Figure 3

Nucleotide sequence of the Arabidopsis At-IE gene and the amino acid sequence predicted from the At-IE cDNA. Nucleotide number refers to the A (+1) of the first ATG in the ORF. The putative polyadenylation signal and TTTGTA motif are double underlined. The vertical arrow indicates the polyadenylation site. Possible TATA and CAAT elements and a putative GCN4-recognition element (GCRE) in the At-IE promoter region are underlined.

Figure 4

Alignment of the amino acid sequence predicted from the Arabidopsis At-IE cDNA and the corresponding proteins of microbial origins. Ec, E. coli (accession no. X13462; Carlomagno et al., 1988); Sy, Synechocystis sp. PCC6803 (accession no. D90917; Kaneko et al., 1996); Rs, Rhodobacter sphaeroides (accession nos. X87256 and X82010; Oriol et al., 1996); Mj, Methanococcus jannaschii (accession nos. U67484 and U67585; Bult et al., 1996); Sc, S. cerevisiae (accession no. J01331; Donahue et al., 1982). Asterisks show the stop codon and dashes inserted to maximize the alignment. Residues conserved among all of the compared sequences are shaded.

Figure 5

RNA-blot analysis of the At-IE mRNA levels. Lane 1, One-week-old plants; lane 2, roots from 2-week-old plants; lane 3, leaves from 2-week-old plants; lane 4, roots from 3-week-old plants; lane 5, leaves from 3-week-old plants; lane 6, roots from 4-week-old plants; lane 7, leave from 4-week-old plants; lane 8, siliques from 4-week-old plants. Membrane was hybridized with a ³²P-labeled PstI-EcoRV fragment of the At-IE cDNA. Total RNA (10 μg) prepared from Arabidopsis seedlings was electrophoresed in each lane. The photograph of the ethidium bromide-stained gel for the blotting is also shown at the bottom of the RNA-blot analysis.

Figure 6

Genomic Southern-blot analysis. Genomic DNA (10 μg) was prepared from Arabidopsis leaves and was digested with restriction enzymes (B, BamHI; Bg, BglII; E, EcoRI; Hc, HincII; and Xb, XbaI). Hybridization was performed using a ³²P-labeled PstI-EcoRV fragment of the At-IE cDNA. The λ-DNA digested with HindIII is shown as a molecular size marker.

Figure 7

Complementation of the hisI mutation of strain UTH903 with putative functional domains of PRA-PH and PRA-CH. A, Exon-intron relationship between the At-IE gene structure and the At-IE cDNA for the bifunctional PRA-PH:PRA-CH protein is shown schematically. B, Expression plasmids were designed to contain the putative catalytic domains and used for the complementation assay for the His auxotrophy of the E. coli hisI mutant. The symbols + and − indicate the ability and inability, respectively, of the plasmids to suppress the E. coli UTH903hisI mutation. E. coli UTH903 cells were transformed with pKF347, representing the full-length of the At-IE cDNA; pKF372 carrying the full-length insert truncated in its putative chloroplast transit sequence; pKF371, corresponding to the N-terminal segment; pKF362 for the N-terminal segment without the putative chloroplast transit sequence; or pKF363 for the C-terminal half of the At-IE protein. After transformation, cells were plated onto M9-Glc minimal agar plates. The portion of the putative chloroplast transit sequence is shaded.

Figure 8

At-IE-dependent AICAR production determined in the assay mixture containing S. cerevisiae HIS1, the hisA protein of E. coli, and one of the expressed recombinant proteins. The At-IE protein without the putative chloroplast transit peptide, the putative PRA-PH (hisE), and the putative PRA-CH (hisI) domains were expressed as the fusion proteins with a maltose-binding protein using a pMAL-c2 bacterial expression vector. The expression vectors used were the same as those presented in Figure 7.