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Originally published In Press as doi:10.1074/jbc.M702662200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30827-30835, October 19, 2007
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Phenylalanine Biosynthesis in Arabidopsis thaliana

IDENTIFICATION AND CHARACTERIZATION OF AROGENATE DEHYDRATASES*Formula

Man-Ho Cho{ddagger}, Oliver R. A. Corea{ddagger}§, Hong Yang{ddagger}, Diana L. Bedgar{ddagger}, Dhrubojyoti D. Laskar{ddagger}, Aldwin M. Anterola{ddagger}, Frances Anne Moog-Anterola{ddagger}, Rebecca L. Hood§, Susanne E. Kohalmi§, Mark A. Bernards§, ChulHee Kang, Laurence B. Davin{ddagger}, and Norman G. Lewis{ddagger}1

From the {ddagger}Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, the §Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada, and the School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660

Received for publication, March 28, 2007 , and in revised form, August 28, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had kcat/Km values of 1050, 7650, and 1560 M-1 s-1 for arogenate versus 38, 240, and 16 M-1 s-1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had kcat/Km values of 1140, 490, and 620 M-1 s-1, with prephenate not serving as a substrate unless excess recombinant protein (>150 µg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1–ADT6.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The aromatic amino acid Phe, formed in both plants and microorganisms, serves as a building block for proteins and as a pathway intermediate to a wide range of aromatic compounds. For mammals, Phe is also an essential dietary requirement. In vascular plants, some of the physiologically important secondary metabolites derived from Phe include pigments and defense molecules (e.g. flavonoids, proanthocyanidins, oligomeric lignans, cyanidins, etc.) (1–3), phytoalexins (e.g. isoflavones) (4), UV protectants (5), as well as the structural lignin and suberin biopolymers (6–8). Taken together, the metabolism of Phe in planta can account for up to 30–40% of organic carbon depending upon the species (9).

In principle, prephenate (from the shikimate-chorismate pathway (10–12)) can be converted into Phe via either phenylpyruvate with subsequent transamination, and/or via arogenate by transamination of prephenate followed by dehydration/decarboxylation (Fig. 1). With microbes, two different enzymes, prephenate dehydratase (PDT)2 and arogenate dehydratase (ADT), are able to catalyze the dehydration/decarboxylation reactions of prephenate and arogenate to afford phenylpyruvate and Phe, respectively. Moreover, whereas most known microorganisms contain PDTs (13–15), an arogenate-specific pathway has also been described in Pseudomonas diminuta and P. vesicularis (16). A third class of enzyme, cyclohexadienyl dehydratase (CDT), common in enteric bacteria, can also either utilize prephenate or arogenate as a substrate (17, 18). In addition, some microorganisms contain dual function "P-proteins" with a N-terminal chorismate mutase domain followed by a PDT domain (19).

Microbial PDTs are often allosterically regulated by binding of pathway end products (19), this being envisaged to occur through a C-terminal ACT domain (name derived from aspartokinase, chorismate mutase and TyrA), whereas CDTs are not (18). The P-proteins also contain a C-terminal ACT domain that has been shown to bind Phe (19). As a result of these different enzyme types, some bacteria, such as Pseudomonas aeruginosa, can use both routes to Phe (20), whereas others, such as Escherichia coli, only utilize phenylpyruvate (21).

Accordingly vascular plants might be anticipated to employ either one or both of these pathways for Phe biosynthesis. Indeed, in the mid-1980s, two research groups reported ADT activities using partially purified enzyme preparations from Nicotiana silvestris cell cultures and spinach (Spinacia oleracea) chloroplasts (22), as well as from etiolated seedlings of Sorghum bicolor (23). Since then, arogenate has been provisionally considered as an intermediate in Phe biosynthesis in plants (7, 9, 22–27). In agreement with this, a prephenate aminotransferase activity was described in some plant species, such as N. silvestris (28) and etiolated shoots of S. bicolor (29). It has not, however, been possible to detect arogenate in the study of various extracts from a number of vascular plants (26) under the conditions employed, indicating that it must be present (at best) at very low levels. This, however, is quite frequently observed in primary metabolism.


Figure 1
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  		FIGURE 1.
Proposed biosynthetic pathway from prephenate and arogenate to Phe in plants and microorganisms.

 
Attempts to establish the presence of PDT activity in higher plants, by comparison, have either been inconclusive (30) (in pea) or unsuccessful (22) (using both N. silvestris and spinach). Yet a recent report indicated that Phe nevertheless results from the activity of PDTs in Arabidopsis (31). Thus it remains unclear in the literature whether Phe results from ADT and/or PDT conversions in plants, and it has become essential to biochemically characterize the corresponding dehydratases involved.

In The Arabidopsis Information Resource data base, there are six Arabidopsis genes (At1g11790, At3g07630, At2g27820, At3g44720, At5g22630, and At1g08250), putatively annotated as PDT homologues. This annotation is based solely on sequence comparison (and thus a reliance on existing annotations) of bacterial sequences. However, because there is no existing functional linkage between any ADT enzyme activity and the corresponding gene sequence, none are annotated as being arogenate dehydratase-like. Yet recently, Ehlting et al. (27) assigned ADT labels (ADT1–ADT6, respectively) to all six Arabidopsis PDT-like genes, but also without any functional characterization in support of this nomenclature. To investigate whether any of these genes encode functional ADTs, their coding sequences were cloned with the corresponding functional recombinant proteins expressed in E. coli. Although all six displayed ADT activity, only ADT1, ADT2, and ADT6 showed any level of PDT activity, albeit at much lower levels related to ADT. This study describes the biochemical parameters obtained for each recombinant protein and provides the first functional characterization of ADTs, the phenylalanineforming machinery, in Arabidopsis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Instrumentation—HPLC analyses employed an Alliance 2695 HPLC system (Waters, Milford, MA) equipped with either a NovaPak C18 column (150 x 3.9 mm; Waters), or a Pico-Tag column (300 x 3.9 mm; Waters). Protein purification was carried out on a fast protein liquid chromatography (Amersham Biosciences) system.

Materials—Phenylmethylsulfonyl fluoride and reagents for making buffers were bought from Sigma; phenylisothiocyanate was from Pierce. PD-10 and DEAE-Sepharose were purchased from GE Healthcare, with POROS MC 20 obtained from Applied Biosystems and Dowex 21K Cl- and AG1-X8 Cl- from Bio-Rad. The expression vector, pET43.1 Ek/LIC, the RosettaTM (DE3) E. coli cells and BugBuster protein extraction reagent were purchased from Novagen. SuperScriptTM II reverse transcriptase, BL21 StarTM (DE3) E. coli cells, and TRIzol reagent were procured from Invitrogen, whereas rDNase I (DNA-free) was from Ambion and Fast RNA Pro-Green Kit from QBioGene. PicoTag reversed phase column (150 x 3.9 mm inner diameter) and PicoTag sample diluent were purchased from Waters (Milford, MA). Prephenate (~78% purity) was obtained from Sigma and purified (>95% purity) using a Chromabond C18 cartridge (Macherey-Nagel) eluted with HPLC grade water. Crude arogenate was isolated from Neurospora crassa (75001/5212/C-167) cultures as described in Ref. 32, then purified (33), lyophilized, and stored at -80 °C until needed.

Cloning of Arabidopsis ADT/PDT—Clones U16103 [GenBank] (for At1g11790) and U11052 [GenBank] (for Atg07630) were obtained from the Arabidopsis Biological Research Center, with the open reading frames of each gene individually amplified and subcloned into the expression vector, pET43.1 Ek/LIC, which was designed for cloning and high level expression of peptide sequences fused with the Nus·TagTM protein. Primers used for subcloning were 5'-GACGACGACAAGATGGCTCTGAGGTGT-3' and 5'-GAGGAGAAGCCCGGTTTATCTGACTAGATC-3' for At1g11790 and 5'-GACGACGACAAGATGGCAATGCACACTGTTCGA-3' and 5'-GAGGAGAAGCCCGGTTTAGAGCATTGTAGTGTC-3' for At3g07630, respectively. The remaining ADTs/PDTs (At2g27820, At3g44720, At5g22630, and At1g08250) were individually amplified from an Arabidopsis cDNA library and subcloned into the expression vector, pET44 Ek/LIC. Primers used for subcloning were 5'-GACGACGACAAGATGCGTGTAGCTTATCAAGG-3' and 5'-GAGGAGAAGCCCGGTCACAATGAAAATGTTG-3' for At2g27820, 5'-GACGACGACAAGATGCGTGTAGCTTACCAAG-3' and 5'-GAGGAGAAGCCCGGTTCATGCTTCTTCTGTG-3' for At3g44720, 5'-GACGACGACAAGATGCGTGTCGCGTATCAAG-3' and 5'-GAGGAGAAGCCCGGTTCATACGTCTTCGCTAG-3' for At5g22630, and 5'-GACGACGACAAGATGCGCGTCGCTTATCAAG-3' and 5'-GAGGAGAAGCCCGGTTACGATGAAGTTGATG-3' for At1g08250, respectively. Recombinant plasmids named pADT1–pADT6, bearing open reading frames of At1g11790, At3g07630, At2g27820, At3g44720, At5g22630, and At1g-08250, respectively, were transformed into Gigasingles competent cells, according to the manufacturer's instructions (Novagen). After sequence confirmation, pADT1–pADT6 were individually transformed into either BL21 StarTM (DE3) (pADT1) or RosettaTM (DE3) (pADT2-pADT6) E. coli cells for expression of recombinant proteins.

Tissue Preparation and RNA Isolation—Leaf, stem, floral, and whole silique tissues were harvested from at least 4-week-old (flowering) wild type Arabidopsis thaliana plants (The Ara-bidopsis Information Resource data base accession number CS3879, ecotype Columbia) grown in sterile soil under 24-h fluorescent light (150 µE/m2/s) at 20 °C. For root tissues, the plants were initially grown on 0.5x Murashige and Skoog basal medium with Gamborg's vitamins (MS; Sigma) and then transferred into 1x MS liquid medium and grown to maturity. The media were supplemented with plant preservative medium (Plant Cell Technology). Upon harvest, all of the tissues were immediately submerged into liquid nitrogen. To isolate total RNA, 100 mg of each plant tissue was lysed in 1 ml of TRIzol containing a large lysing matrix bead and shaken in a FastPrep machine (BIO101/QBioGene) once (flower and root) or twice (leaf, stem, and silique) for 45 s (setting 4). RNA was isolated using the QBioGene Fast RNA ProGreen Kit according to the manufacturer's instructions.

Reverse Transcriptase (RT)-PCR Analysis—RNA (10 µg) was treated with rDNase I (2 units) to remove single- and double-stranded DNA, and 2 µg was reverse transcribed using Super-ScriptTM II reverse transcriptase (200 units). PCR amplification was performed using primer pairs specific to the 3'-untranslated region of each ADT (ADT1-F, 5'-AATTGATTTCATGTTACCATACCG-3'; ADT1-R, 5'-TCTCAACAATGAAGAGTTTGAAGC-3'; ADT2-F, 5'-TCTTCTTGTTTGTGACAGAGATCC-3'; ADT2-R, 5'-AAACTAAACAGAATTTGTAACAAGAGC-3'; ADT3-F, 5'-CAACGTGTGAAGTCAATTGTG-3'; ADT3-R, 5'-CGATCAAACGAAACTCCAAAG-3'; ADT4-F, 5'-TCATTATTCACGTGGTGATTAGG-3'; ADT4-R, 5'-TTTGCTTTTCTTGGTTAAAATCG-3'; ADT5-F, 5'-TCGTTTTGCATGTGAAGTGG-3'; ADT5-R, 5'-TCATCAGAACCAAACTACAAACC-3'; ADT6-F, 5'-TGTAAATTTTGGAGAAGATAACAAAG-3'; and ADT6-R, 5'-TTTACCAATTGTTTATTGATTTATACG-3'). To ensure that PCR products originated solely from mRNA, each template was PCR-amplified using a primer set that specifically recognizes a genomic DNA sequence that is not transcribed (GEN-F, 5'-GCTTTCATGTTTTAGCAATGGCG-3'; GEN-R, 5'-ATTAATTCTTCGTGGATGCCGG-3'). The absence of amplification of a 600-bp product in DNase treated versus untreated samples demonstrates that experimental PCR amplifications are solely based on RNA templates. PCR products were sequenced to ensure that the proper ADT was amplified. As a control, PCR was also performed for all RNA extracts using an RNA-specific primer set (TUB2-F, 5'-TGTCTGCAAGGGTTCCAGGTT-3'; TUB2-R, 5'-TCACCTTCTTCATCCGCAGTT-3') that amplifies the ubiquitously expressed gene TUBULIN2 (TUB2) (34, 35). All PCR amplifications were performed according to the following protocol: 2 min at 94 °C; 35 cycles of 15 s at 94 °C, 1 min at 55 °C, 2 min at 68 °C; and a final extension for 10 min at 68 °C. PCR products were size-separated in a 6% polyacrylamide gel and stained with ethidium bromide.

Expression in E. coli and Purification of Recombinant Nus Proteins—The E. coli transformants, individually harboring pADT1–pADT6, were grown at 37 °C in LB medium supplemented with carbenicillin (0.1 mg ml-1) until an A600 of ~0.9 was reached, at which time isopropyl thio-beta-D-galactoside (0.3 mM) was added. The cells were grown at 16 °C for 20–24 h, individually harvested by centrifugation (3,000 x g for 20 min), and stored at -20 °C until required. The cell pellets were individually resuspended in BugBuster Protein Extraction Reagent according to the manufacturer's instructions, with the cell debris removed by centrifugation (10,000 x g for 20 min). These crude protein preparations were individually applied to a DEAE-Sepharose column (1.6 x 3 cm) equilibrated in buffer A (50 mM potassium phosphate, pH 7.5), and eluted with buffer A containing 1 M NaCl. Fractions from the flow-through were combined and further subjected to POROS MC 20 column (1 x 9 cm) chromatography with Cu2+ (CuSO4) as metal ions (36). After washing with buffer B (20 mM Tris·HCl, pH 7.9, 500 mM NaCl, 50 ml) containing 20 mM imidazole, recombinant proteins were individually eluted with an imidazole gradient (20–500 mM, 100 ml) in buffer B. Fractions containing each recombinant protein (i.e. eluting between 50 and 130 mM imidazole) were combined, buffer-exchanged to Buffer A, and concentrated to a small volume (~0.5 ml) using a Centricon Plus-20 (Amicon). Because of the instability of the ADT proteins, the purification steps described above (from cell harvest to affinity chromatography) were carried out over a 24 h duration, with purified proteins immediately used for enzyme assays.

PDT Assays—PDT activities were determined by measuring phenylpyruvate formation. Each assay consisted of prephenate (1 mM), recombinant protein (15 µg for ADT1 and ADT6, 2 µg for ADT2, and >150 µg for ADT3–ADT5), and buffer A in a total volume of 50 µl. The enzymatic reaction was initiated by individual addition of recombinant protein, and after incubation for 30 min at 37 °C, MeOH (50 µl) was added to stop the reaction. An aliquot (50 µl) of each assay mixture was then subjected to reversed phase HPLC (NovaPak C18, Waters) analysis. Phenylpyruvate and prephenate were separated using a linear MeOH gradient (0–18%) in eluant A (20 mM sodium phosphate, pH 6.9) at a flow rate of 1 ml min-1, with detection at 210 nm. The amount of phenylpyruvate was determined using an external calibration curve.

ADT Assays—ADT activities were measured by determining the amounts of phenylthiocarbamyl derivatives of Phe (26). Each assay mixture consisted of arogenate (1 mM), the recombinant protein (100 ng for ADT1 and ADT3–ADT6 and 50 ng for ADT2) and potassium phosphate buffer (200 mM, pH 7.5) in a total volume of 50 µl. The enzymatic reaction was initiated by individual addition of recombinant protein. After incubation at 37 °C for 30 min, the reaction mixtures were immediately placed on ice to stop the reaction and freeze-dried. Lyophilized reaction mixtures were subsequently individually derivatized with phenylisothiocyanate as previously described (26). After drying in vacuo, the reaction mixtures were reconstituted in 100 µl of PicoTag sample diluent. An aliquot (50 µl) was subjected to HPLC analysis on a PicoTag reversed phase column. The phenylthiocarbamyl derivatives were eluted with a linear gradient (0–100%) of eluant B (CH3CN-MeOH-H2O, 45:15:40) to eluant C (0.07 M sodium acetate, titrated to pH 6.5 with glacial acetic acid-CH3CN, 97.5:2.5) over 20 min at 1 ml min-1. Detection was at 254 nm. Amounts of Phe formed were determined using an external calibration curve. Control assays were conducted for both arogenate and prephenate activities as described above but in the absence of recombinant proteins.


Figure 2
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  		FIGURE 2.
Domain structure and amino acid sequence alignment of putative plant ADTs/PDTs, bacterial PDTs, and bacterial P-proteins. A, schematic representation of domain arrangements. Putative domains are: ADT/PDT, arogenate/prephenate dehydratase; PDT, prephenate dehydratase; ACT, regulatory domain; TP, (putative) N-terminal transit peptide; CM, chorismate mutase. B, partial sequence alignment using a highly conserved C-terminal portion of the putative ADT/PDT (blue bar at bottom) and the N-terminal portion of the putative ACT domains (green bar at bottom). The boundaries between the two domains are as described for E. coli P-protein by Zhang et al. (19). Amino acids with similar properties are shown in the same color. Amino acid residues that are either identical or similar in ≥66% of sequences are shaded in green or gray, respectively. Additional symbols below the alignment indicate residues identified by mutagenesis to be involved in catalytic activity in either E. coli ({blacktriangleup} and {triangleup}) or C. glutamicum (• and {circ}) (44, 45, 49, 51, 52), as well as the absolutely conserved arginine of the TRF motif ({diamond}). Sequences marked with an asterisk are annotated as having an ACT domain. The numbers on the right-hand side are in reference to the N-terminal end of each protein, i.e. the beginning of the transit peptide (Arabidopsis), chorismate mutase domain (E. coli, Haemophilus influenzae, and P. aeruginosa), or PDT domain (all other species). BjPDT, Bradyrhizobium japonicum, NP_768061; CgPDT, Corynebacterium glutamicum, AAA23304; CtPDT, Chlorobium tepidum, AMM72891; EcPDT, E. coli, ZP_00708880; EfPDT, Enterococcus faecium, EAN09096; HiPDT, H. influenzae, P43900; LlPDT, Lactococcus lactis, AAK05840; MlPDT, Mesorhizobium loti, BAB51940; PaPDT, P. aeruginosa, AAG06554; SpPDT, Streptococcus pneumoniae, AAK75467.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
In Silico Analysis of Putative PDT and ACT Domains in Bacterial and Plant Genes and Proteins—Although ADT activity has been biochemically detected in bacteria, plants, and green algae (16, 22, 23, 37, 38), there are no reports of molecular studies of any putative ADT genes in any organisms, only that of PDTs and CDTs. Our in silico screening, however, resulted in the provisional identification of six putative PDT domain-containing genes in Arabidopsis (At1g08250, At1g11790, At2g27820, At3g07630, At3g44720, and At5g22630) as mentioned above. Additionally, 35 possible ADT/PDT sequences were identified for other plant species, i.e. in Allium cepa, Glycine max, Gossypium hirsutum, Hordeum vulgare, Lotus japonicus, Lycopersicon esculentum, Medicago truncatula, Nicotiana benthamiana, Oryza sativa, Pinus taeda, Saccharum officinarum, Solanum tuberosum, Triticum aestivum, Vitis vinifera, and Zea mays, in available data bases (NCBI and TIGR), based on sequence similarity to the six Arabidopsis genes (nucleotide and translated amino acid sequences). All plant putative ADTs/PDTs contained three distinct (albeit putative) domains at the amino acid level: a N-terminal transit peptide, followed by a PDT-like domain, and a C-terminal ACT or ACT-like domain (Fig. 2A). By contrast, all bacterial PDT sequences lack the N-terminal signal peptide extension, whereas P-proteins have an N-terminal chorismate mutase domain. Although previously only two of the ADT/PDTs were annotated as possessing a partial (At1g11790) or full (At3g07630) ACT domain, our analysis suggests that the remaining four do as well. All share many of the conserved amino acids (Fig. 2B) considered important for feedback regulation in bacteria (see below). However, whether these enzymes utilize prephenate, arogenate, or both as a substrate could not be predicted by sequence analysis alone.

Alignments also indicate that there are many conserved amino acids throughout the putative ADT/PDT domains in the selected plant and bacterial sequences examined (Fig. 2B). However, the rooted phylogenetic tree, constructed from amino acid sequences encompassing ADT/PDT and ACT domains from all species included, suggests that the plant and bacterial sequences are evolutionarily quite distinct from one another (Fig. 3 and supplemental Fig. S1). The plant sequences seem to form three distinct subgroups that are conserved across monocots and dicots. Interestingly, the Arabidopsis genes that segregated into subgroup I (At1g11790) and II (At3g07630) contain introns, whereas the remaining four (At2g27820, At3g44720, At5g22630, and At1g08250), which cluster within a subgroup III, do not (data not shown). Further distinction between Arabidopsis ADTs/PDTs is evident in the amino acid sequence similarities within their catalytic (ADT/PDT) domains. That is to say, all four Arabidopsis ADTs/PDTs from subgroup III show a higher degree of ADT/PDT domain amino acid similarity to each other (81–98%) than to either of the other two ADTs (61–72%) (Table 1). The conservation of these subgroups may denote unique roles (e.g. in distinct metabolic branches) for the members of each group (discussed later).


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  		TABLE 1
Amino acid sequence identity of the PDT domains of putative Arabidopsis ADTs/PDTs The sequences were aligned using ClustalW, and then identity matrices were created using BioEdit, version 7.0.0. The genes are named according to the nomenclature of Ehlting et al. (27).

 
In Vivo Expression of Arabidopsis ADTs/PDTs—It was next important to establish whether any of the ADT/PDT genes were differentially expressed in Arabidopsis tissues (e.g. roots, leaves, stems, flowers, and siliques) of flowering Arabidopsis plants. However, because only two of the six ADT/PDT genomic sequences have introns, we were unable to design primers that span intron-exon borders, as is normally done to create RNA-specific primers. Therefore, to ensure that RT-PCR amplifications solely derived from RNA templates (and not contaminant genomic DNA in our preparations), each isolated RNA sample was treated with rDNase I prior to reverse transcription. The cDNAs from the DNase-treated and untreated samples were then subsequently tested using a primer set complementary to a nontranscribing genomic sequence, which generates a 600-bp PCR product only from genomic DNA. Absence of this 600-bp PCR fragment in DNase I-treated samples confirmed that the samples were completely free from residual contaminating genomic DNA sequences. Only those RNA samples free from residual contaminating genomic DNA were used for the expression analyses. As a further control, TUBULIN2, a constitutively expressed gene (34, 35), was also amplified for each sample. From the data thus obtained, all six ADTs/PDTs appeared to be expressed in all tissues from which RNA was isolated, including roots, leaves, stems, flowers, and siliques (Fig. 4). When comparing results from the different ADTs/PDTs, however, variations in band intensities cannot be interpreted as differences in ADT/PDT expression levels because different primers with potentially different amplification properties were used for each one. Because all ADT/PDT genes appear to be expressed in all tissues examined, it became essential to establish whether all members of the Arabidopsis ADT/PDT family possessed ADT or PDT enzymatic activity, or both, through recombinant protein expression and kinetic analyses.


Figure 3
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  		FIGURE 3.
Phylogenetic relationship between selected plant and bacterial ADTs/PDTs. The rooted phylogenetic tree, generated with DNAMAN using a bootstrap of 1000, compares amino acid sequences containing both ADT/PDT and ACT domains. The numbers at the branch points give the boot-strapping values. The horizontal scale indicates sequence divergence. The clustering of bacterial sequences into Gram-positive and Gram-negative groups as well as plant ADT/PDT subgroups are delineated with dashed lines. The CDT sequence for P. aeruginosa is included as a representative of this group. Bacterial sequences used are as in the Fig. 2 legend as well as PaCDT, P. aeruginosa, AAC08596 and SpoPDT Schizosaccharomyces pombe, CAB10811. One O. sativa sequence (Os07g32774), which does not appear to contain an ACT domain, was omitted from the analysis. A more complete phylogenetic tree, which includes an additional ~40 plant and bacterial sequences, is presented in the supplemental material.

 


Figure 4
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  		FIGURE 4.
RT-PCR analyses of Arabidopsis ADTs. mRNA for RT-PCR analysis was collected from mature flowering plants as described under "Experimental Procedures." PCR products were size-separated in a 6% polyacrylamide gel and stained with ethidium bromide. R, root; L, leaf; S, stem; Si, silique; F, flower. The amplification products for ADTs and TUB2 can be distinguished by size: ADT1, 146 bp; ADT2, 135 bp; ADT3, 145 bp; ADT4 176 bp; ADT5, 117 bp; ADT6, 103 bp; TUB2, 204 bp. M, 100-bp ladder. The 200- and 100-bp markers are shown for all ADTs, whereas for TUB2 the 200- and 300-bp markers are visible.

 
Expression of Recombinant ADT/PDT Proteins—Each ADT was thus successfully expressed as a Nus-fused recombinant protein in E. coli in soluble His-tagged form using the pET 43.1 EK/Lic vector. This approach was necessary to overcome general inclusion body problems (39–42) that were initially encountered when many other attempts to express His-tagged proteins all failed. Each protein was then individually purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, and Cu2+ affinity chromatography, respectively (data not shown); all manipulations including assays were carried out within 24 h. Following SDS-PAGE analyses, the apparent molecular mass of each was estimated to be ~110 kDa, in good agreement with the deduced molecular mass of the Nus-fused ADT/PDTs (data not shown).

Arabidopsis ADT/PDTs Encode Functionally Competent ADTs—Individual enzyme assays performed using either arogenate or prephenate as potential substrates indicated that the six recombinant ADT/PDT homologues were catalytically active, i.e. all six gene products encoded a functional dehydratase. For detailed kinetic studies, enzyme reactions were carried out at pH 7.5, because arogenate and prephenate are unstable under acidic conditions and can be spontaneously converted into Phe and phenylpyruvate, respectively (43). The recombinant At1g11790, At3g07630, and At1g08250 proteins all had relatively similar affinities for both arogenate and prephenate (Km values ranging from 0.80 to 3.05 mM for arogenate and 0.68 to 2.44 mM for prephenate, respectively; Table 2). These Km values are greater than previously reported ADT activities in S. bicolor (23), Erwinia herbicola (17), and P. aeruginosa (18), which ranged from 0.09 to 0.32 mM and PDT activities in E. herbicola (17), P. aeruginosa (18), E. coli (19, 44), Mycobacterium tuberculosis (15), and Corynebacterium glutamicum (45), which ranged from 0.07 to 0.56 mM. The higher Km values for the ADTs may be due to the presence of the Nus tag, because fusion tags have been shown to affect the Km of other recombinant proteins (46). On the other hand, the calculated maximal velocities (Vmax) of recombinant At1g11790, At3g07630, and At1g08250 were significantly higher for arogenate (31, 60.6, and 42.61 pkat µg-1 protein) than for prephenate (0.28, 1.6, and 0.4 pkat µg-1 protein) (Table 2). Moreover, the overall catalytic efficiencies (kcat/Km) for arogenate were ~28-, 32-, and 98-fold higher than that of prephenate (Table 2). These data indicate that these three gene products are more ADT-like rather than PDT-like, and we thus provisionally designate At1g11790, At3g07630, and At1g08250 as ADTs (ADT1, ADT2, and ADT6, respectively, using the nomenclature of Ehtling et al. (27)).


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  		TABLE 2
Kinetic parameters for the arogenate and prephenate dehydratase activities of recombinant protein derived from six Arabidopsis ADT/PDT genes

 
The remaining three gene products (At2g27820, At3g44720, and At5g22630) also displayed a substrate preference for arogenate with Km values ranging from 0.43 to 10.08 mM and Vmax values from 52.32 to 5.17 pkat µg-1 protein. The catalytic efficiency (kcat/Km) of At2g27820 (1140 M-1 s-1; Table 2) was similar to that of ADT1 and ADT6; the kcat/Km values of At3g44720 and At5g22630 were lower (e.g. 490 and 620 M-1 s-1, respectively; Table 2). More striking, however, was the nearly complete lack of any detectable PDT activity of recombinant proteins derived from these three proteins (Table 2), with only minute levels of activity detected at a very high protein concentration (>150 µg). Thus these three gene products are also provisionally designated as the functional ADTs, i.e. ADT3, ADT4, and ADT5 (27).

Because these enzymes are the first functional ADTs to be individually expressed and assayed, it was also important to demonstrate that the conditions for the prephenate and arogenate assays did not favor ADT over PDT activity. Therefore, a previously characterized PDT from Methanocaldococcus jannaschii (courtesy of Dr. Peter Kast) was assayed using the same conditions, with 0.2 µg of enzyme in each reaction. The Km and kcat values obtained for prephenate were somewhat lower than that reported by Kleeb et al. (47): 370 versus 22 µM and 8.9 versus 12.3 s-1, respectively. In our hands, the differences in Km and kcat were possibly due to the widely different assay conditions used. However, the arogenate activity of PDT from M. jannaschii was not previously tested. We found that ADT activity was virtually absent when using 0.2 µg of enzyme in each sample, and a minute conversion to Phe was only observed with the addition of excess (5–10 µg) enzyme.

These data are in stark contrast to those of Warpeha et al. (31), who reported an indirect prephenate conversion into phenylpyruvate in crude protein extracts from wild type Arabidopsis but not PD1 mutants (deficient in At2g27820; ADT3). These authors did not, however, test arogenate conversion into Phe, precluding any comparison between PDT and ADT activities. Our ADT3 Nus fusion protein, however, had essentially no PDT activity in vitro while displaying substantial ADT activity. It is possible, however, that there may be some residual PDT activity in the native protein. This does not seem likely though because ADT1, but not ADT3, was able to complement a PDT-deficient yeast mutant (pha2) (48).3

Secondary Structure, Mode of Catalysis, and Regulation Predictions—Despite the low degree of identity noted earlier between bacterial and plant sequences, their corresponding ADT/PDT domains seem all to be composed of five or six {alpha}-helices as suggested by secondary structure predictions (data not shown). In addition, many of the specific catalytic sites identified in bacterial PDTs were conserved in the Arabidopsis homologues. Specifically, residues important for PDT catalysis in E. coli (Asn160, Ser208, Gln215, and Thr278 (44); Fig. 2B, {blacktriangleup}) and C. glutamicum (Glu64, Ser99, and Thr183 (45); Fig. 2B, •), were also present in Arabidopsis ADTs). Moreover, the Thr identified in each of these is part of a TRF motif that might be involved in either catalytic activity or substrate binding (44, 45, 49). Indeed, even though there are no crystal structures for ADTs/PDTs from any species, this well characterized TRF motif is predicted to be in an {alpha}-helix. Furthermore, although CDTs have little apparent sequence similarity to ADTs/PDTs (see below), they nevertheless also have a Thr at this motif (18), perhaps indicative of its importance for enzyme function. The arginine in the TRF motif (Fig. 2B, {diamond}) is also absolutely conserved across the 70 plant and bacterial species so far surveyed (i.e. the same as those in supplemental Fig. S1). Conservation of these amino acid residues in Arabidopsis ADTs thus also support the hypothesis that these enzymes potentially function as aromatic ring-forming dehydratases.

Sequence similarity within the ACT domain may suggest similar functions, because it is the three-dimensional structure of this domain that is important for feedback regulation (for review, see Ref. 50). Indeed, sequence alignments with Phe hydroxylase, together with mutational analysis and isothermal titration calorimetry of the E. coli P-protein, identified two candidate regulatory regions predicted to be involved in Phe binding and feedback inhibition, i.e. 309GALV312 and 329ESRP332, respectively (Fig. 2B, {triangleup}) (51, 52). The ESRP motif, which is the most hydrophilic region in the P-protein regulatory domain, is predicted to be part of a loop and is conserved across most ADT/PDT sequences examined, including all six Arabidopsis ADTs (Fig. 2B). On the other hand, the GALV motif, which is the most hydrophobic region of the regulatory domain of the P-protein, is very likely part of a secondary structural element ({alpha}-helix). However, it is only partially conserved across these same sequences, with Gly and Leu having the highest degree of conservation across bacterial and plant ADTs/PDTs. Meanwhile, another residue, Trp388 (not shown), which is also proposed to be involved in Phe binding (37), is not conserved; instead, it is Leu, Ile, or Val in Arabidopsis ADTs. Furthermore, mutational analysis of C. glutamicum PDT identified two more amino acids in the ACT domain, Arg202 and Gly224, that caused resistance to Phe-mediated feedback inhibition of PDT activity (53). These amino acids were conserved as Lys and Ala, respectively, in many other bacterial PDTs, as well as in the six Arabidopsis ADTs (Fig. 2B, {circ}). In summary, the high degree of similarity within the ACT domain of Arabidopsis ADTs and the high number of conserved residues with bacterial PDTs provisionally suggest that all six Arabidopsis ADT genes could possess functional ACT domains.

Finally, when the protein sequence of the CDT from P. aeruginosa was compared with that of the ADTs/PDTs from both plants and bacteria, it clearly resided as an outlier (Fig. 3), suggesting little sequence similarity. This is in agreement with earlier reports that CDTs are distinct from bacterial PDTs (18) and confirms that they are even more distantly related to putative plant ADTs/PDTs. There were no homologues for CDT-like proteins found in the Arabidopsis genome.

Concluding Remarks—Although it has been proposed that vascular plants preferentially form Phe via arogenate (22, 23), no direct evidence, e.g. purification of an ADT, cloning of an ADT-encoding gene, and/or expression of a functionally competent recombinant protein, has been reported. In this study, we cloned all six putative Arabidopsis ADTs/PDTs predicted as possibly involved in Phe biosynthesis. Interestingly, all six appeared to be expressed throughout the plant, preventing strict assignment of individual isoforms to specific cellular processes at this time. Future work will therefore determine to what extent the ADTs are differentially expressed in the various cell types of each tissue at various points of growth/development, using, for example, quantitative real time PCR.

All six genes were subsequently expressed in active recombinant form in E. coli, and each was shown to preferentially catalyze the decarboxylative dehydration of arogenate to afford Phe (Table 2). Three of these, ADT1, ADT2, and ADT6, were also able to catalyze the decarboxylation/dehydration of prephenate directly, whereas the remaining three had an apparent strict requirement for arogenate. Our biochemical studies also indicated that the ADT activities were >28 times higher than that of PDT (Table 2), where PDT activity was even detected, providing support that Arabidopsis preferentially utilizes arogenate in Phe biosynthesis. Phylogenetic analysis places the six Arabidopsis ADTs into three distinct subgroups (Fig. 3 and supplemental Fig. S1). The three ADTs studied herein that showed both ADT and PDT activity were distributed across all three subgroups, whereas the remaining three were restricted to subgroup III. In future, more extensive studies will be needed both in vitro (e.g. to investigate feedback analysis of all six Arabidopsis ADTs, and site-directed mutagenesis targeting ADT and ACT domain residues), as well as in vivo (e.g. gene knock-out, expression) studies. These are necessary to fully understand the last steps of Phe biosynthesis and the involvement of each isoform (e.g. in the various possible metabolic pathways derived from Phe). Other detailed studies will also be directed toward establishing the catalytic mechanism of ADTs and their three-dimensional structures.


   FOOTNOTES
 
* This work was supported in part by National Science Foundation Grant MCB-0417291, United States Dept. of Energy Grant DE-FG-0397ER20259, National Institutes of Health Grant GM66173, and funds from the G. Thomas and Anita Hargrove Center for Plant Genomic Research and the Natural Sciences and Engineering Research Council of Canada (to M. A. B. and S. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed. Tel.: 509-335-8382; Fax: 509-335-8206; E-mail: lewisn{at}wsu.edu.

2 The abbreviations used are: PDT, prephenate dehydratase; ADT, arogenate dehydratase; CDT, cyclohexadienyl dehydratase; PITC, phenylisothiocyanate; HPLC, high pressure liquid chromatography; RT, reverse transcriptase. Back

3 O. R. A. Corea, M. A. Bernards, and S. E. Kohalmi, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Peter Kast for the M. jannaschii PDT.



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 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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