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Plant Physiol. (1998) 118: 275-283
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
Ko Fujimori and
Daisaku Ohta*
Takarazuka Research Institute, Novartis Pharma K.K., 10-66 Miyuki-cho, Takarazuka 665-8666, Japan (K.F.); and 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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INTRODUCTION |
Biochemistry and genetics of His biosynthesis (Fig.
1) have been extensively studied in a
number of microorganisms (Winkler, 1987 ; Alifano et al., 1996). In
eubacteria such as Escherichia coli and Salmonella
typhimurium, the complete nucleotide sequences of the His operons
have been determined (Carlomagno et al., 1988), and it was shown that
eight structural genes are organized in a single operon encoding all of
the enzymes catalyzing the 11 steps of the pathway (Carlomagno et al.,
1988). In Lactococcus lactis the His biosynthetic genes
appeared to be clustered in an operon containing several ORFs of
unknown function (Delorme et al., 1992), whereas in archaebacteria
such as Methanococcus vannielii and Methanococcus
jannaschii, these genes are scattered throughout the chromosome
(Beckler and Reeve, 1986; Bult et al., 1996).
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| 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.
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The complete genomic nucleotide sequence of Synechocystis
sp. PCC6803 has recently been determined, and it was found that His
biosynthetic genes do not organize an operon (Kaneko et al., 1996). In
lower eukaryotes, including Saccharomyces cerevisiae, the
His biosynthetic genes are found in different loci (Mortimer et al.,
1994). It has also been established that several of these genes encode
multifunctional enzymes (Alifano et al., 1996): hisIE encodes PRA-CH and PRA-PH, hisB codes for
imidazoleglycerolphosphate dehydratase and histidinolphosphate
phosphatase, and hisD codes for histidinol dehydrogenase in
E. coli and S. typhimurium (Carlomagno et al.,
1988). However, the hisI and hisE reactions in Azospirillum brasilense (Fani et al., 1993) and also in some archaebacteria (Beckler and Reeve, 1986, Bult et al., 1996) are catalyzed by separate
protein molecules. On the other hand, multifunctional enzymes with the
activities corresponding to the hisIE and hisD proteins are encoded by
HIS4 in S. cerevisiae (Donahue et al., 1982) and
Pichia pastoris (Crane and Gould, 1994), the
his7+ gene of Schizosaccharomyces
pombe (Apolinario et al., 1993), and the his-3 gene of
Neurospora crassa (Legerton and Yanofsky, 1985).
Genetic analysis of yeast his4 mutants suggested that the HIS4 protein can be divided into three subdomains, HIS4A, HIS4B, and
HIS4C, which correspond to hisI, hisE, and hisD, respectively (Donahue
et al., 1982).
In the past several years we have isolated cDNAs encoding enzymes
involved in higher-plant His biosynthesis. They are the histidinol
dehydrogenase from Brassica oleracea (Nagai et
al., 1991) and the imidazoleglycerolphosphate dehydratase from
Arabidopsis and wheat (Tada et al., 1994). Here we report
the isolation of an Arabidopsis cDNA that encodes a bifunctional
protein (At-IE) that has both PRA-PH and PRA-CH activities through
genetic complementation of an E. coli hisI mutant defective
in the PRA-CH activity. Furthermore, we isolated and characterized a
single-copy gene coding for the At-IE protein. Analysis of the
At-IE gene and recombinant enzyme expression studies
revealed that the N-terminal and the C-terminal halves of the At-IE
protein correspond to PRA-CH and PRA-PH, respectively.
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MATERIALS AND METHODS |
Plant Materials, Bacterial Strains, and Culture Media
Arabidopsis ecotype Columbia seedlings (Col-0, Lehle Seeds,
Tucson, AZ) were germinated on germination medium 0.8% (w/v)
agar plates (Valvekens et al., 1988) under sterile conditions, and the
seedlings were cultivated in a growth chamber maintained at 23°C and
80% RH with a 16-h light/8-h dark cycle with the light intensity of
150 µE. Bacterial strains used in this study are listed in
Table I. Escherichia coli
JM109 strain was used as the host for the propagation and manipulation
of plasmid DNA. Luria-Bertani medium and M9 minimal medium for E. coli were prepared as described previously (Sambrook et al.,
1989).
Isolation of an Arabidopsis cDNA Encoding PRA-CH (hisI) and PRA-PH
(hisE)
A cDNA library of 7-d-old Arabidopsis seedlings (Mizutani et al.,
1997) was converted to a phagemid stock by in vivo excision according
to the manufacturer's instructions (Stratagene). E. coli
UTH903 cells were transformed with 5 µg of the cDNA library phagemid
stock (1.9 × 105 cells/µg plasmid). The
transformation mixture was plated on M9 minimal plates containing 0.2%
(w/v) Glc supplemented with 100 µg mL 1
ampicillin, 25 µg mL1 streptomycin, and an
amino acid mixture without L-His (Sambrook et al., 1989),
and incubated at 37°C for 2 d. Plasmids were recovered from
purified his+ colonies and tested for their
ability to suppress the His auxotrophy of strain UTH903. The clone
containing the longest insert (pKF323 = pAt-IE) was identified
after restriction-enzyme analysis, and its DNA sequence was completely
determined.
DNA Sequencing
Nucleotide sequences were determined from both strands by the
dideoxy chain-termination method (Sanger et al., 1977) using a dye
terminator cycle sequencing kit (PRISM, Applied Biosystems). Nucleotide
and amino acid sequences were analyzed using DNASIS version 3.4 software (Hitachi Software Engineering Co., Yokohama, Japan) and by
performing the BLAST search (Altschul et al., 1990) of the National
Center for Biotechnology Information.
Nucleic Acid Hybridization Analysis
Genomic DNA (10 µg) was prepared from 4-week-old Arabidopsis
seedlings as described previously (Sambrook et al., 1989). After digestion with the restriction enzymes, DNA fragments were separated electrophoretically in a 0.7% (w/v) agarose gel and transferred to a
Hybond N+ nylon membrane (Amersham) in 0.4 N NaOH (Sambrook et al., 1989). Hybridization was performed
using the At-IE cDNA as a probe at 37°C overnight in a solution
containing 40% (v/v) formamide, 5× Denhardt's solution, 6× SSC,
0.5% (w/v) SDS, and 100 µg mL1 sheared
salmon-sperm DNA (Sigma; Sambrook et al., 1989). The membrane was
washed twice in 2× SSC/0.1% (w/v) SDS at room temperature for 10 min
and then twice in 0.5× SSC/0.1% (w/v) SDS at 50°C for 15 min. Blots
were exposed to a Hyperfilm-MP (Amersham) for 2 d at
 80°C using an intensifying screen.
For RNA-blot analysis, total RNA was prepared as described previously
(Lagrimini et al., 1987), and 10-µg aliquots of the sample were
electrophoretically separated in a 2.2 M formaldehyde-1.2% (w/v) agarose gel in Mops buffer (Sambrook et al., 1989) and then transferred to a Hybond N+ nylon membrane in 6×
SSC. The At-IE cDNA was labeled by the random-priming method (Feinberg
and Vogelstein, 1983) using [ -32P]dCTP.
Hybridization was carried out at 42°C overnight in a solution consisting of 50% (v/v) formamide, 5× Denhardt's solution, 6× SSC,
0.5% (w/v) SDS, and 100 µg mL1 sheared
salmon-sperm DNA (Sambrook et al., 1989). The blots were washed twice
in 2× SSC/0.1% (w/v) SDS at room temperature for 10 min and twice in
0.2× SSC/0.1% (w/v) SDS at 55°C for 15 min. Blots were analyzed
using a bioimaging analyzer (BAS2000, Fuji Photo Film Co., Tokyo,
Japan).
Isolation of the At-IE Gene of
Arabidopsis
A genomic clone containing a fragment of the
At-IE gene was identified by screening approximately
5 × 105 plaques of an Arabidopsis genomic
DNA library made with  ZAPII (Stratagene). Plaques were transferred
to Colony/Plaque Screen nylon membranes (NEN) and hybridized overnight
at 42°C with the full-length At-IE cDNA as a probe. Membranes were
washed once in 2× SSC/0.1% (w/v) SDS at room temperature for 30 min,
once in 0.5× SSC/0.1% (w/v) SDS at 50°C for 30 min, and once in
0.2× SSC/0.1% (w/v) SDS at 50°C for 30 min. The blots were then
exposed to Hyperfilm-MP films for 16 h. Positive plaques were
rescreened until pure phages were obtained.
Expression of the At-IE cDNA in E. coli
For heterologous expression experiments, DNA fragments encoding
different domains of the At-IE protein were amplified by PCR using
specific sets of primers (Table I) and the At-IE cDNA as a template.
The PCR products were double digested with BamHI and XhoI and cloned into a
BamHI-SalI-digested pMAL-c2 vector (New England
Biolabs, Inc., Beverly, MA) to obtain expression plasmids (Table I). A
set of PRI28 and PRI30 was used for the amplification of a 931-bp
fragment containing the entire coding region (pKF347); PRI44 and PRI30
for a 778-bp fragment encoding the At-IE protein without the region
corresponding to the putative chloroplast transit peptide (pKF372);
PRI28 and PRI45 for a 495-bp fragment for an N-terminal segment
(pKF371); PRI44 and PRI45 for a 342-bp fragment for an N-terminal
domain without the putative chloroplast transit peptide (pKF362); and
PRI46 and PRI30 for a 436-bp fragment of a C-terminal segment (pKF363).
The expressed recombinant fusion proteins were purified by amylose
resin affinity-column chromatography (New England Biolabs.). These
expression plasmids were also used to transform E. coli BL21
and UTH903 cells.
Complementation of the E. coli hisI
Mutant
Strain UTH903 was transformed with either pAt-IE (= pKF323)
or an empty pBluescript SK() (pBS SK[]). pAt-IE (= pKF323)
contains a full-length Arabidopsis hisIE cDNA. After the
transformation, strain UTH903 harboring either an empty pBluescript or
pAt-IE was cultivated overnight in M9-Glc medium supplemented with 1 mM histidinol at 37°C. After harvesting, E. coli cells were homogenized in 50 mM potassium
phosphate (pH 7.5) buffer by mild sonication. After centrifugation at
10,000g for 10 min, the soluble fraction was passed
through a Sephadex G-25 column (PD-10, Pharmacia). AICAR production was
determined photometrically by measuring A550 by the Bratton-Marshall method (Ames et al., 1961). AICAR of 10 µM solution gave an A550 of
0.270 (Ames et al., 1961) in a 1-cm light path. Protein was assayed by
the Bradford (1976) method.
Enzyme Assay
Recombinant proteins corresponding to the putative functional
domains were produced as described above and used in a coupling enzyme
assay (Ames et al., 1961) to mimic the His biosynthetic pathway (Fig.
1). This coupling assay was used because neither the substrate nor the
reaction product of the At-IE was available. The reaction mixture (170 µL) contained 111 mM Tris-Cl (pH 8.5), 22.2 mM MgCl2, 83.3 mM KCl,
5.6 mM ATP (Sigma), the recombinant proteins to be assayed,
and a recombinant HIS1 protein of Saccharomyces cerevisiae
and a recombinant hisA protein of E. coli as the coupling enzymes. These recombinant HIS1 and hisA proteins were produced in
E. coli as fusion proteins with the maltose-binding protein using a pMAL-c2 vector (New England Biolabs) and affinity purified using amylose resin columns (New England Biolabs). The reaction was
started by the addition of 10 µL of 10 mM PRPP (Sigma),
and the reaction mixture was incubated at 30°C for 15 min. In this assay system, BBM III was produced from ATP and PRPP according to the
His biosynthetic scheme (Fig. 1) through the activities of the HIS1
(the first step of His pathway), Arabidopsis At-IE (the second and
third steps), and the hisA (the fourth step) proteins. The BBM III
produced was hydrolyzed to AICAR in HCl at 95°C. AICAR was determined
as described above.
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RESULTS |
Cloning of an Arabidopsis cDNA That Suppresses an E. coli
hisI Mutation
An Arabidopsis cDNA encoding a bifunctional enzyme with PRA-PH and
PRA-CH activities was isolated through genetic complementation of a
bacterial His auxotrophic mutant. Thus, an E. coli hisI
defective mutant (UTH903) was transformed with a phagemid library
prepared from 7-d-old Arabidopsis seedlings, and 20 prototrophic
colonies of 9.5 × 105 transformants were
identified after cultivating for 2 d on M9 minimal agar plates.
Upon retransformation of strain UTH903, 16 of the isolated 20 plasmids
were found to be able to suppress the His auxotrophy. The DNA inserts
of these 16 plasmids exhibited identical restriction patterns, and DNA
sequencing showed that they were derived from the same cDNA fragment
(data not shown). The remaining four of the identified His prototrophic
colonies might be revertants, since their plasmids contained DNA
inserts of inconsistent nucleotide sequences and failed to recomplement the His auxotrophy (data not shown). One of the plasmids (pKF323 = pAt-IE) containing the longest insert was sequenced completely and used
for further analyses.
Strain UTH903 was transformed with either pAt-IE or an empty
pBluescript, and crude cell extracts were prepared for an AICAR production assay. The AICAR production observed with the cells transformed with pAt-IE (2.06 ± 0.19 nmol
mg1 protein min1) was
comparable to that with XL1-Blue as a control, whereas no AICAR
production was detected with the UTH903 transformed with an empty
pBluescript. These results were consistent with the complementation experiments (Fig. 2) in which the UTH903
transformed with pAt-IE was able to grow on M9-Glc minimum medium but
no bacterial growth was observed when transformed with a pBluescript
empty vector.
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| 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.
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The At-IE cDNA contained an ORF of 843 bp encoding a polypeptide of 281 amino acids with a calculated molecular mass of 31,666 D (Figs.
3 and 4).
Nucleotide sequence analysis showed that the consensus motif
surrounding a translation initiation codon (AACAATGGC) in
plants (Lütcke et al., 1987) was well conserved as
TAAAATGGC in the At-IE cDNA. Several consensus sequences
required for the correct 3 end formation of transcripts in plants were
also found in the 3 untranslated region (Fig. 3). Thus, a putative
polyadenylation signal sequence, AATAAA (Wahle and Keller,
1992), was found 23 bp upstream from the adenylation tail. The TTTGTA
motif, which is considered to be involved in the stability of
transcripts (Rothnie et al., 1994), was also identified at position
+2363 (Fig. 3).
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| 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.
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| 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.
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The predicted primary structure of the At-IE protein was compared with
those of microorganisms available in the nucleotide database (Fig. 4).
Sequence alignment indicated that an N-terminal segment (spanning
residues Gly-65 to Phe-158) of the At-IE protein was highly homologous
to the conserved region among the hisI proteins so far reported and
that the C-terminal region encompassing residues Leu-179 to Arg-267 was
homologous to the domain conserved among the microbial hisE proteins
(Fig. 4). These results indicate that the At-IE cDNA encoded a
bifunctional protein of PRA-CH and PRA-PH, of which domain organization
has also been found in the PRA-CH (hisI) and PRA-PH (hisE) enzymes of
eubacteria and lower eukaryotes but not in archaebacteria. This
putative domain structure of the At-IE protein was investigated through
both the gene structure analysis and the recombinant protein expression
studies, as described later. It was also found that the N-terminal
portion of approximately 50 amino acids showed no significant homology
to those of the hisIE proteins from microorganisms but showed the
properties characteristic of chloroplast transit peptides (von Heijne
and Nishikawa, 1991).
RNA-Blot Analysis
Upon northern-blot analysis, the transcript size of the
At-IE gene appeared to be approximately 1.2 kb (Fig.
5), which was in good agreement with the
predicted size from the At-IE cDNA (Fig. 3). The At-IE gene
was expressed ubiquitously in plants throughout development. The
highest expression level for the At-IE mRNA was observed in roots of
3-week-old plants and in inflorescence stems of 4-week-old plants.
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| 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.
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Cloning and Sequencing of an At-IE Genomic Clone
To examine the number of the At-IE genes in
Arabidopsis, Southern-blot analysis was performed using the full-length
At-IE cDNA as a probe. The digestion with HincII, which cuts
once within the cDNA, gave rise to two bands (Fig.
6). BglII also has a single restriction site, but the digestion yielded a single hybridization signal that we thought was derived from two bands overlapping. The
digestion with either BamHI, EcoRI, or
XbaI, of which no restriction sites were found in the cDNA
sequence, yielded only a single hybridization signal even after long
exposure (data not shown). These hybridization patterns indicated that
the At-IE gene exists as a single copy in the genome of
Arabidopsis.
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| 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 -DNA digested with
HindIII is shown as a molecular size marker.
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The At-IE gene was cloned by screening an Arabidopsis
genomic library using the At-IE cDNA as a probe, and a phagemid
harboring a 5.5-kb EcoRI-EcoRI fragment was
identified to contain the At-IE gene (Figs. 3 and
7). The At-IE gene consists of
five exons divided by four introns (Figs. 3 and 7). Intron-splice sites
of all of the introns follow the "GU-AG" rule, which is observed at
intron-splice sites of all eukaryotic organisms, including higher
plants (Breathnach and Chambon, 1981; Simpson and Filipowicz, 1996).
Amino acid sequence alignment (Figs. 3 and 4) showed that intron 1 was
located at the junction of the putative transit peptide portion encoded
by exon 1 and the PRA-CH (hisI) domain encoded by exons 2 and 3. It was
also found that intron 3 was located at the putative boundary between
the PRA-CH (hisI) domain and the PRA-PH (hisE) domain, which was
encoded by exons 4 and 5. Two homologous regions among the bacterial
hisIE proteins so far reported were also found to be conserved in the
At-IE protein (Fig. 4).
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| 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.
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In the 5 untranslated region several putative regulatory elements were
found (Fig. 3). Possible TATA and CAAT elements were identified at
positions 166 and 195, respectively. Furthermore, a sequence motif,
TAACTC, similar to the S. cerevisiae GCN4-responsive element, GCRE (Arndt and Fink, 1986), was located at position 288 (Fig. 3).
Characterization of the Domain Structure of the At-IE Protein
Sequence comparison (Fig. 4) suggested that the At-IE protein
molecule consisted of a putative chloroplast transit peptide and two
separate catalytic domains corresponding to the PRA-CH (hisI) and
PRA-PH (hisE) proteins, respectively. This overall putative domain
structure of the At-IE protein was confirmed through heterologous
expression studies.
Both pKF347 and pKF372 were able to suppress the His auxotrophy of
E. coli strain UTH903 (Fig. 7). The insert of pKF372 encoded an At-IE protein of which the N-terminal extension had been truncated (Fig. 7). Therefore, the successful suppression of the His auxotrophy of UTH903 with pKF372 indicated that the N-terminal extension was not
essential for the catalytic activity, supporting the idea that this
N-terminal extension corresponded to a chloroplast transit peptide. The
expressed recombinant protein using pKF372 (Fig. 7) was enough to
support the AICAR production in the assay system containing the
recombinant HIS1 protein of S. cerevisiae and the hisA
protein of E. coli (Fig. 8).
Thus, pKF372 contained the cDNA that encodes a protein catalyzing the
hisIE reactions (Fig. 1). However, the recombinant protein prepared
with pKF362 (the N-terminal domain), which was able to complement the
hisI mutation of UTH903 (Fig. 7), did not work for the AICAR
production (Fig. 8). Thus, the N-terminal domain catalyzed the hisI
(PRA-CH) reaction but did not have hisE (PRA-PH) activity. The cDNA
insert of pKF363 was derived from exons 4 and 5 coding for the
C-terminal domain (Fig. 7). This plasmid failed to suppress the His
auxotrophy of UTH903 (Fig. 7) and could not support the AICAR
production (Fig. 8), indicating that the C-terminal domain did not
catalyze the hisI (PRA-CH) reaction but was involved in the hisE
(PRA-PH) reaction. On the other hand, the AICAR production was
reconstituted when the recombinant proteins encoded by pKF362 and
pKF363 were mixed in the reaction mixture (Fig. 8), indicating that the
C-terminal domain corresponded to the hisE (PRA-CH) domain. No
histidinol dehydrogenase activity was observed with the protein
produced with pKF347 (data not shown).
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| 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.
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These results have demonstrated that the single-copy gene, the
At-IE gene of Arabidopsis, encodes the bifunctional protein of which the N-terminal and C-terminal domains separately catalyze the
two successive reactions of PRA-CH (hisI) and PRA-PH (hisE), respectively, in the His biosynthetic pathway. Also, it was found that
the At-IE protein, like most bacterial enzymes, was not accompanied by
a histidinol dehydrogenase domain, which is encoded by HIS4 in S. cerevisiae (Donahue et al., 1982).
| |
DISCUSSION |
A number of eukaryotic genes, including those of plant origin,
have been isolated by performing heterologous genetic complementation of E. coli or S. cerevisiae mutants (Minet et
al., 1992; Senecoff and Meagher, 1993; Tada et al., 1994). This method
was also successful in isolating the At-IE cDNA from Arabidopsis with
the use of strain UTH903 defective in hisI (PRA-CH) activity, which
encodes a bifunctional protein with hisI (PRA-CH) and hisE (PRA-PH)
activities. The amino acid sequence predicted from the At-IE cDNA is
significantly homologous to both the hisIE proteins of eubacteria and
their counterparts in yeasts and fungi. It has been reported that the
hisI and hisE domains are located at the N-terminal and the C-terminal
halves, respectively, of the microbial hisIE proteins (Donahue et al., 1982; Fig. 4). In the At-IE protein, the region encompassing residues Gly-65 to Phe-158 is homologous to the bacterial hisI and S. cerevisiae HIS4A proteins with PRA-CH activity, and the second
region from Leu-179 to Arg-267 is homologous to the bacterial hisE and
the HIS4B of S. cerevisiae with PRA-PH activity.
These two functional domains for hisI (PRA-CH) and hisE (PRA-PH) were
able to be separately expressed, with the corresponding cDNA fragments
retaining their individual catalytic activities (Figs. 7 and 8). Thus,
the At-IE protein molecule is shown to be composed of two independent
functional domains connected by a gap region, which shows no
significant homology with other corresponding proteins (Fig. 7). It was
also demonstrated that the At-IE protein does not contain the
histidinol dehydrogenase domain encoded by HIS4 of S. cerevisiae. It has been shown already that histidinol dehydrogenase is encoded by a separate gene in higher plants (Nagai et
al., 1993).
Genomic Southern-blot analysis showed that the At-IE exists
as a single-copy gene consisting of five exons divided by four introns,
encoding the three putative functional units, the chloroplast transit
peptide (exon 1), the PRA-CH domain (exons 2 and 3), and the PRA-PH
domain (exons 4 and 5). This organization suggests that the gene for
the chloroplast transit peptide and the genes for the protein
corresponding to PRA-CH and PRA-PH have fused during evolution.
Examples for similar gene organization containing an intron between a
putative chloroplast transit peptide and a mature protein have also
been reported for a few nuclear genes encoding chloroplastic proteins
(Wolter et al., 1988; Gantt et al., 1991). It is thought that the
sequences for the chloroplast transit peptide have been attached to the
genes for chloroplastic proteins during the process of gene transfer
from the chloroplast to the nucleus (Gantt et al., 1991). The yeast
HIS4 and fungal his3 proteins also have N-terminal leader
sequences of unknown function, whereas no significant N-terminal
homology was seen between the At-IE protein and these microbial
proteins.
It has been demonstrated that other higher-plant His biosynthetic
enzymes, imidazoleglycerolphosphate dehydratase (Tada et al., 1995) and
histidinol dehydrogenase (Nagai et al., 1993), are localized in
chloroplasts. Furthermore, the cDNAs encoding N-[(5-PR)-formimino]-5-aminoimidazole-4-carboxamide
ribonucleotide isomerase and Gln amidotransferase/cyclase of
Arabidopsis have also been shown to contain the regions corresponding
to putative N-terminal transit peptides (K. Fujimori and D. Ohta,
unpublished results). It is therefore possible that the entire His
biosynthesis may be completed in chloroplasts, whereas the first enzyme
of the His pathway, ATP-PR transferase, has not been characterized yet.
His biosynthesis is an extremely energy-consuming process, which
requires 41 ATP molecules for each His molecule produced (Alifano et
al., 1996). In other words, the compartmentalization of the His pathway
in chloroplasts is favorable to ensure efficient energy supply. At
least six genes encoding eight steps of His biosynthesis in plants
exhibited constitutive expression patterns throughout development, and
there were no clear tissue-specific expression patterns (Nagai et al.,
1993; Tada et al., 1995; K. Fujimori and D. Ohta, unpublished results).
In microorganisms, His biosynthesis is regulated through controlled
gene expression and the feedback regulation of ATP-PR transferase
activity by L-His (Alifano et al., 1996). Isolation and
characterization of ATP-PR transferase are essential for understanding
the mechanism that regulates higher-plant His biosynthesis.
| |
FOOTNOTES |
*
Corresponding author; e-mail ohtad{at}orange.ocn.ne.jp; fax
81-866-56-9454.
Received March 10, 1998;
accepted June 16, 1998.
The nucleotide sequence data reported in this paper will appear in the
nucleotide sequence databases with the accession nos. AB006082 (At-IE
cDNA) and AB006083 (At-IE gene).
| |
ABBREVIATIONS |
Abbreviations:
AICAR, 5-aminoimidazole-4-carboxamide-1- -D-ribofuranoside.
BBM
III, N-[(5-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide
ribonucleotide.
ORF, open reading frame.
PR, phosphoribosyl.
PRA-CH, phosphoribosyl-AMP cyclohydrolase.
PRA-PH, phosphoribosyl-ATP
pyrophosphohydrolase.
PRPP, phosphoribosyl pyrophosphate.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Nobuko Uodome for skillful
experimental assistance.
| |
LITERATURE CITED |
Alifano P,
Fani R,
Liò P,
Lazcano A,
Bazzicalupo M,
Carlomagno MS,
Bruni CB
(1996)
Histidine biosynthetic pathway and genes: structure, regulation, and evolution.
Microbiol Rev
60:
44-69
[Free Full Text]
Altschul SF,
Gish W,
Miller W,
Myers EW,
Lipman DJ
(1990)
Basic local alignment search tool.
J Mol Biol
215:
403-410
[CrossRef][ISI][Medline]
Ames BN,
Martin RG,
Garry BJ
(1961)
The first step of histidine biosynthesis.
J Biol Chem
236:
2019-2026
[Free Full Text]
Apolinario E,
Nocero M,
Jin M,
Hoffman CS
(1993)
Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies.
Curr Genet
24:
491-495
[CrossRef][ISI][Medline]
Arndt K,
Fink GR
(1986)
GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5 TGACTC 3 sequences.
Proc Natl Acad Sci USA
83:
8516-8520
[Abstract/Free Full Text]
Beckler GS,
Reeve JN
(1986)
Conservation of primary structure in the hisI gene of the archaebacterium, Methanococcus vannielli, the eubacterium Escherichia coli, and the eucaryote Saccharomyces cerevisiae.
Mol Gen Genet
204:
133-140
[CrossRef][Medline]
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Breathnach R,
Chambon P
(1981)
Organization of eucaryotic split genes coding for proteins.
Annu Rev Biochem
50:
349-383
[CrossRef][ISI][Medline]
Bult CJ,
White O,
Olsen GJ,
Zhou L,
Fleischmann RD,
Sutton GG,
Blake JA,
FitzGerald LM,
Clayton RA,
Gocayne JD,
and others
(1996)
Science
273:
1058-1073
[Abstract]
Carlomagno MS,
Chiariotti L,
Alifano P,
Nappo AG,
Bruni CB
(1988)
Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.
J Mol Biol
203:
585-606
[CrossRef][ISI][Medline]
Crane DI,
Gould SJ
(1994)
The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids.
Curr Genet
26:
443-450
[CrossRef][Medline]
Delorme C,
Ehrlich SD,
Renault P
(1992)
Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.
J Bactriol
174:
6571-6579
[Abstract/Free Full Text]
Donahue TF,
Farabaugh PJ,
Fink GR
(1982)
The nucleotide sequence of the HIS4 region of yeast.
Gene
18:
47-59
[CrossRef][ISI][Medline]
Fani R,
Alifano P,
Allotta G,
Bazzicalupo M,
Carlomagno MS,
Gallori E,
Rivellini F,
Polsinelli M
(1993)
The histidine operon of Azospirillum brasilense: organization, nucleotide sequence and functional analysis.
Res Microbiol
144:
187-200
[Medline]
Feinberg AP,
Vogelstein B
(1983)
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem
132:
6-13
[CrossRef][ISI][Medline]
Gantt JS,
Baldauf SL,
Calie PJ,
Weeden NF,
Palmer JD
(1991)
Transfer of rpl22 to the nucleus greatly preceded its loss from the chloroplast and involved the gain of an intron.
EMBO J
10:
3073-3078
[ISI][Medline]
Kaneko T,
Sato S,
Kotani H,
Tanaka A,
Asamizu E,
Nakamura Y,
Miyajima N,
Hirosawa M,
Sugiura M,
Sasamoto S,
and others
(1996)
Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.
DNA Res
3:
109-136
[Abstract]
Lagrimini LM,
Burkhart W,
Moyer M,
Rothstein S
(1987)
Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: molecular analysis and tissue-specific expression.
Proc Natl Acad Sci USA
84:
7542-7546
[Abstract/Free Full Text]
Legerton TL,
Yanofsky C
(1985)
Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa.
Gene
39:
129-140
[CrossRef][Medline]
Lütcke HA,
Chow KC,
Mickel FS,
Moss KA,
Kern HF,
Scheele GA
(1987)
Selection of AUG initiation codons differs in plants and animals.
EMBO J
6:
43-48
[ISI][Medline]
Minet M,
Dufour M-E,
Lacroute F
(1992)
Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs.
Plant J
2:
417-422
[ISI][Medline]
Mizutani M,
Ohta D,
Sato R
(1997)
Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta.
Plant Physiol
113:
755-763
[Abstract]
Mortimer RK,
Romano P,
Suzzi G,
Polsinelli M
(1994)
Genome renewal: a new phenomenon revealed from a genetic study of 43 strains of S. cerevisiae derived from natural fermentation of grape musts.
Yeast
10:
1543-1552
[CrossRef][ISI][Medline]
Nagai A, Suzuki K, Ward E, Moyer M, Mano J, Beck J, Tada S, Hashimoto
M, Chang J-Y, Ryals J, and others (1993) Histidinol dehydrogenase
in higher plants. Purification, cloning and expression. In
Research in Photosynthesis, Vol IV. Kluwer Academic Publishers,
Dordrecht, The Netherlands, pp 95-98
Nagai A,
Ward E,
Beck J,
Tada S,
Chang J-Y,
Scheidegger A,
Ryals J
(1991)
Structural and functional conservation of histidinol dehydrogenase between plants and microbes.
Proc Natl Acad Sci USA
88:
4133-4137
[Abstract/Free Full Text]
Oriol E,
Méndez-Alvarez S,
Barbé J,
Gilbert I
(1996)
Cloning of the Rhodobacter sphaeroides hisI gene: unifunctionality of the encoded protein and lack of linkage to other his genes.
Microbiology
142:
2071-2078
[Abstract]
Rothnie HM,
Reid J,
Hohn T
(1994)
The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3-end formation in plants.
EMBO J
13:
2200-2210
[ISI][Medline]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sanger F,
Nicklen S,
Coulson AR
(1977)
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:
5463-5467
[Abstract/Free Full Text]
Senecoff JF,
Meagher RB
(1993)
Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants.
Plant Physiol
102:
387-399
[Abstract]
Simpson GG,
Filipowicz W
(1996)
Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organization of the spliceosomal machinery.
Plant Mol Biol
32:
1-41
[CrossRef][ISI][Medline]
Tada S,
Hatano M,
Nakayama Y,
Volrath S,
Guyer D,
Ward E,
Ohta D
(1995)
Insect cell expression of recombinant imidazole glycerolphosphate dehydratase of Arabidopsis and wheat and inhibition by triazole herbicides.
Plant Physiol
109:
153-159
[Abstract]
Tada S,
Volrath S,
Guyer D,
Scheidegger A,
Ryals J,
Ohta D,
Ward E
(1994)
Plant Physiol
105:
579-583
[Abstract]
Valvekens D,
Montagu MV,
Lusebettens MV
(1988)
Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.
Proc Natl Acad Sci USA
85:
5536-5540
[Abstract/Free Full Text]
von Heijne G,
Nishikawa K
(1991)
Chloroplast transit peptides: the perfect random coil?
FEBS Lett
278:
1-3
[CrossRef][ISI][Medline]
Wahle E,
Keller W
(1992)
The biochemistry of 3-end cleavage and polyadenylation of messenger RNA precursors.
Annu Rev Biochem
61:
419-440
[ISI][Medline]
Winkler ME (1987) Biosynthesis of histidine. In FC
Neidhardt, JL Ingraham, KB Low, B Magasanik, M Schaechter, HE Umbarger,
eds, Escherichia coli and Salmonella typhimurium.
Cellular and Molecular Biology, Vol I. American Society of
Microbiology, Washington, DC, pp 395-411
Wolter FP,
Fritz CC,
Willmitzer L,
Schell J,
Schreier PH
(1988)
rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.
Proc Natl Acad Sci USA
85:
846-850
[Abstract/Free Full Text]
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Abstract
Introduction