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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

An LL-Diaminopimelate Aminotransferase Defines a Novel Variant of the Lysine Biosynthesis Pathway in Plants^1,[W]

Biotech Center and Department of Plant Biology and Pathology, Rutgers University, New Brunswick, New Jersey 08901 (A.O.H., T.L.); Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 (C.G.); and BASF Plant Science, Research Triangle Park, North Carolina 27709 (B.K.S.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Although lysine (Lys) biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate, the available evidence suggests that none of the known DAP-pathway variants found in nature occur in plants. A new Lys biosynthesis pathway has been identified in Arabidopsis (Arabidopsis thaliana) that utilizes a novel transaminase that specifically catalyzes the interconversion of tetrahydrodipicolinate and LL-diaminopimelate, a reaction requiring three enzymes in the DAP-pathway variant found in Escherichia coli. The LL-DAP aminotransferase encoded by locus At4g33680 was able to complement the dapD and dapE mutants of E. coli. This result, in conjunction with the kinetic properties and substrate specificity of the enzyme, indicated that LL-DAP aminotransferase functions in the Lys biosynthetic direction under in vivo conditions. Orthologs of At4g33680 were identified in all the cyanobacterial species whose genomes have been sequenced. The Synechocystis sp. ortholog encoded by locus sll0480 showed the same functional properties as At4g33680. These results demonstrate that the Lys biosynthesis pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.

View larger version (22K): [in this window] [in a new window]   Figure 1. The mechanisms for DAP/Lys synthesis. The pathways labeled in the diagram include two variants that use either succinylCoA or acetylCoA. Another uses Ddh to directly convert THDPA to m-DAP. The enzyme reported in this article, LL-DAP-AT, directly converts THDPA to LL-DAP. The DAP dehydrogenase and LL-DAP-AT diagrams show only the enzymatic step that differentiates these pathways from the acyl-DAP pathways. Acronyms in the diagram include THDPA, L-2,3,4,5-tetrahydrodipicolinate; LL-DAP, LL-2,5-diaminopimelate; m-DAP, m-2,6-diaminopimelate; DapA, dihydrodipicolninate synthase; DapB, dihydrodipicolinate reductase; DapD, THDPA acyltransferase; DapC, N-acyl-L-2-amino-6-oxopimelate aminotransferase; DapE, N-acyl-LL-2,6-diaminopimelate deacylase; DapF, DAP epimerase; and LysA, m-DAP decarboxylase. The structures of the intermediates are shown on the left.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification of an LL-DAP-AT Activity in Plants

To search for an LL-DAP-AT activity in plants, an assay was developed to measure the production of THDPA using OAB, a compound that yields a dihyrodoquinazolinium adduct that has an absorbance maximum at A₄₄₀. When a soluble extract from axenically grown Arabidopsis plants was incubated with LL-DAP and 2-oxoglutarate (2-OG) as an amino acceptor, a linear formation of 440-nm absorbing material was observed over a period of 90 min (Fig. 2A ). The rate of the reaction was directly proportional to the amount of protein extract added (Fig. 2B). No activity was observed if extract was omitted or when either LL-DAP or 2-OG were absent from the reaction. If the extract was heated in a boiling water bath for 5 min, the activity was completely destroyed. All of these observations strongly suggested that the activity was enzymatic. Moreover, since the source material was axenically grown, the activity must have been derived from the Arabidopsis rather than a contaminating microorganism.

View larger version (13K): [in this window] [in a new window]   Figure 2. LL-DAP-AT activity in Arabidopsis. Graph A shows the time course of a complete reaction with 500 µg protein from Arabidopsis leaf extract (black circles), compared with a reaction lacking LL-DAP (white circles). Graph B shows the relationship between reaction rate and the amount of protein added to the reaction. The 1-mL assay contained 100 µmol HEPESKOH (pH 7.6), 0.5 µmol amino donor, 2.0 µmol 2-OG, and 1.25 mg OAB. The reaction was incubated at 30°C. The protein extract was prepared from Arabidopsis leaves by grinding in liquid nitrogen with 100 mM HEPESKOH (pH 7.6), centrifugation at 10,000g for 15 min, and buffer exchange using an Amicon Ultra 30,000 MWCO filter.

Identification of the Arabidopsis Gene Encoding LL-DAP-AT

Since the characterization of LL-DAP-AT would be greatly facilitated if the gene encoding this enzyme could be identified, a search was conducted of the Arabidopsis genomic loci encoding known and hypothetical aminotransferases. Specific sequence motifs have been defined that would allow aminotransferase genes to be readily identified in the DNA sequence databases. Using these characters, 44 likely aminotransferases were annotated in Arabidopsis (Liepman and Olsen, 2004). Of these, 19 were reported to be uncharacterized, in the sense that the specific reaction catalyzed by the gene product had not yet been established. Since LL-DAP-AT is very likely to be plastid localized, an initial focus was placed on five uncharacterized aminotransferases predicted to be localized to chloroplasts (At1g77670, At2g13810, At2g22250, At4g33680, and At5g57850). The corresponding cDNAs were expressed in Escherichia coli and an extract of each culture was assayed. In this way At4g33680 was identified as a genetic source of LL-DAP-AT activity. None of the other genes produced such an activity.

At4g33680 was annotated as a 461-amino acid, class I/II family aminotransferase. The first 36 amino acids were predicted by TargetP to be a transit peptide for localization of the protein to plastids. The closest paralog to At4g33680 in Arabidopsis is At2g13810, with which it shares 64.4% amino acid identity (Liepman and Olsen, 2004). Despite the homology, recombinant At2g13810 protein did not show LL-DAP-AT activity. It is important to emphasize that there are a number of explanations for why At2g13810 may not have shown LL-DAP-AT activity, but this question has not been explored yet.

Quantitative Enzyme Assays and Properties of the Pure LL-DAP-AT

The kinetic properties of the pure recombinant At4g33680 enzyme were studied using several different assays. The expression and purification of LL-DAP-AT is shown in Figure 3 . The SDS-PAGE analysis shows that the At4g33680 expression plasmid produces a 51-kD protein, identical to the predicted molecular mass of the recombinant protein, and it is purified by nickel-affinity chromatography. The pure enzyme was found to have a 420-nm absorbance feature (data not shown) typically found in enzymes that have pyridoxal phosphate linked to a conserved Lys residue. Most aminotransferases require pyridoxal phosphate as a cofactor (Liepman and Olsen, 2004). The Lys residue at position 305 in the At4g33680 protein is predicted to be the pyridoxal phosphate ligand.

View larger version (53K): [in this window] [in a new window]   Figure 3. Recombinant expression and purification of LL-DAP-AT. E. coli strain BL21-Codon Plus-RIPL carrying pET30b-AtDAT was grown and expression from the plasmid induced as described in "Materials and Methods." The gel shows the profile of 10 µg soluble proteins in uninduced cells compared to induced cells. Also shown is 0.5 µg overexpressed LL-DAP-AT purified by nickel-affinity chromatography. The SDS-PAGE gel contained 12.5% (w/v) acrylamide and was stained with Coomassie Blue.

To examine the activity of LL-DAP-AT, the OAB assay was not ideal because the extinction coefficient of the dihyrodoquinazolinium adduct that OAB forms with THDPA was unknown and the assay would not be useful to determine whether LL-DAP-AT is able to function in the physiologically relevant direction. For this reason quantitative coupled assays were developed to assess the enzyme activity in both the reverse and forward directions. In the reverse direction the formation of THDPA by LL-DAP-AT was measured by coupling with Ddh from Corynebacterium glutamicum, which oxidizes NADPH when converting THDPA to m-DAP. The reaction sequence is shown in Scheme 1.

(1)

Using this coupled-assay system, LL-DAP-AT was found to have an activity of 22.3 µmol min^–1 mg^–1 protein and apparent K_m values of 67 µM for LL-DAP and 8.7 mM for 2-OG (Table III).

(2)

Due to the interference that NADPH formation would have on measurement of NADH produced by the 2-OG dehydrogenase reaction, it was necessary to replace NAD⁺ with thio-NAD⁺. Thio-NADH has an absorbance maximum at 398 nm, which can be discerned from NADPH, which has an absorbance maximum at 340 nm. Figure 4 illustrates such a reaction. The black symbols show the progress of the THDPA-generating reaction monitored at 340 nm. Two plots are shown using 0.55 and 0.055 mM m-DAP. At the completion of the prereaction, the wavelength was changed to 398 nm, 2-OG dehydrogenase was added, and the absorbance monitored for 2 min. The decrease in absorbance when the wavelength was changed to 398 nm indicated that NADPH does not absorb light significantly at this wavelength. The unchanging absorbance after 2-OG dehydrogenase addition indicated that thio-NAD⁺ was not reduced by any combination of the substrates or the enzymes in the mixture. However, after addition of LL-DAP-AT there was a progressive increase in absorbance (gray symbols). The rate of the increase was dependent on the initial concentration of THDPA, which was formed from m-DAP in the prereaction. The results show that LL-DAP-AT can operate in the forward direction. Using this assay system, the specific activity of the LL-DAP-AT was found to be 0.38 µmol min^–1 mg^–1 protein, and it showed an apparent K_m of 38 µM for THDPA and 1.9 mM for Glu (Table III). In total, the kinetic constants confirmed that LL-DAP-AT can catalyze the interconversion of THDPA and LL-DAP in vitro.

View larger version (19K): [in this window] [in a new window]   Figure 4. LL-DAP synthesis activity. The forward assay was conducted in two steps. In the first, THDPA was synthesized from m-DAP using CtDdh. The aminotransferase was assayed in the second step. The prereaction contained in 1 mL 100 µmol HEPESKOH (pH 7.5), 0.5 µmol NADP+, 0.5 µmol (black circle) or 0.05 µmol (black square) m-DAP, 0.3 µmol thio-NAD+, 0.3 µmol CoA, 0.5 µmol Glu, and 32 µg Ddh. The reaction was incubated at 22°C and the increase in A340 was recorded as a measure of m-DAP to THDPA conversion. Then 200 µg of 2-OG dehydrogenase (0.625 µmol min–1 mg–1 protein) was added to the reactions with 0.5 µmol (gray circle) or 0.05 µmol (gray square) m-DAP followed by pure LL-DAP-AT, and the increase in A398 was measured to calculate the activity of the aminotransferase.

View larger version (43K): [in this window] [in a new window]   Figure 5. Complementation of dap mutants with At4g33680. A, Strains AT980 (dapD), AT984 (dapE), and AOH1 (dapD/dapE) were transformed with either the plasmid vector (pBAD33) or with the At4g33680 expression plasmid (pBAD33-AtDAT). Colonies were selected on LB medium with 50 µg mL–1 DAP and 34 µg mL–1 chloramphenicol. Individual colonies were then replica plated onto NZY medium supplemented with 0.2% (w/v) Ara without or with 50 µg mL–1 DAP. The cultures were grown at 30°C for 48 h. B, Diagram of the DAP pathway in E. coli with the reaction catalyzed by LL-DAP-AT indicated. The structure on the left is of THDPA and on the right of LL-DAP.

To assess the taxonomic distribution of LL-DAP-AT, the coding sequence of At4g33680 was used to search the protein sequence databases. A neighbor-joining tree showing the relationship of homologous sequences in plant and cyanobacteria is depicted in Figure 6 . For the sake of comparison the DapC sequences from Bordetella parapertussis, C. glutamicum, and the ArgD sequences from E. coli, Bordetella pertussis, and Bacillus subtilis were included in the analysis. Both DapC and ArgD have been shown to catalyze aminotransfer to N-succinyl-L-2-amino-6-oxopimelate, which is the reaction in the acyl DAP pathways (Ledwidge and Blanchard, 1999; Fuchs et al., 2000; Hartmann et al., 2003) analogous to that catalyzed by LL-DAP-AT (Fig. 1). The tree shows three major clades. One includes the orthologs of LL-DAP-AT, which branches into closely related cyanobacterial and eukaryotic forms. Another includes DapC orthologs, and a third includes ArgD orthologs. The clades share low sequence homology. For example, Synechocystis sll0480 shares about 19% sequence identity with either C. glutamicum or B. parapertussis DapC. In contrast, the most divergent members of the LL-DAP-AT group share a minimum of 46% identity. By way of comparison, the closest sll0480 homologs in C. glutamicum (encoded by locus NCgl0780) and B. parapertussis (encoded by locus BPP2478) show about 23% identity with sll0480. Thus, despite the fact that class I and II family aminotransferases are descended from a common ancestor, the divergence indicates that all the LL-DAP-AT share a distinct lineage that is different from either DapC or ArgD.

View larger version (26K): [in this window] [in a new window]   Figure 6. Phylogenetic tree showing the relationship between LL-DAP-AT orthologs and DapC and ArgD orthologs. The protein sequences were aligned using ClustalW and the neighbor-joining tree was constructed using the program MEGA2 version 2.1 (Kumar et al., 2001). The GenBank accession number or locus tag for the protein sequences used to produce the alignment were Avar03004417, At4g33680, NP_389004.1, NP_882054.1, BPP1996, AAO12273.1, Cwat03005178, CMN323C, Q8X4S6, AAU93923.1, Npun02008059, AY338235.1, Pro-1655, syc0687, SYNW2147, sll0480, tll2102, and Tery02000376. The protein from Thalassiosira pseudonana was deduced from gene model grail.111.8.1 on scaffold_111 obtained from the Web site http://genome.jgi-psf.org/thaps1/thaps1.home.html. The protein from Medicago truncatula was deduced from the sequence between nucleotides 105403 and 110280 of bac clone mth2-36a23 (GenBank accession AC124214). The protein from Physcomitrella patens was deduced from contigs 10435 and 10436 obtained from http://moss.nibb.ac.jp/.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The existence of a novel variant of the DAP pathway was predicted in Arabidopsis based on the finding that this species does not contain an apparent ortholog of DapD nor functional homologs of DapC and DapE (Hudson et al., 2005). Since these enzymes form the core of the prokaryotic acyl pathway for Lys synthesis, their absence and the previous demonstration that a number of plants do not contain Ddh (Chatterjee et al., 1994) raised the question that prompted this study: How do plants bridge the metabolic gap between THDPA and LL-DAP? The findings reported here answer this question. They do so with a single enzyme, an aminotransferase that directly converts THDPA to LL-DAP. This finding adds yet another example to the list of natural variants of the DAP pathway.

LL-DAP-AT is able to bypass the acylation and deacylation steps found in most bacteria. To our knowledge, the function of acylation in the biosynthesis of DAP has never been clearly delineated. The equilibrium between the cyclic and acyclic structures favors THDPA, yet it is the acyclic form that contains the keto group needed for transamination. For this reason it was proposed that acylation speeds the conversion of the ring-structured THDPA to the acyclic form (Berges et al., 1986). The DapD enzyme was envisioned as adding water to the imine of THDPA to produce a trans-piperidine dicarboxylate intermediate to which the acyl group is added, thereby facilitating ring opening. Therefore, in species with an LL-DAP-AT pathway, the rate of Lys synthesis would depend on the spontaneous rate of ring opening unless the process was catalyzed. Whether LL-DAP-AT catalyzes THDPA ring opening remains to be investigated.

Much evidence exists supporting the idea that chloroplasts were derived from an endosymbiosis between a cyanobacterium and a heterotrophic, mitochondrion-containing eukaryote (Falkowski et al., 2004). After the symbiosis the cyanobacterial genes were subsequently transferred to the host nucleus, where they acquired the sequences necessary to target the proteins to the chloroplast (Martin et al., 2002). The conservation and taxonomic distribution of the LL-DAP-AT in eukaryotic photoautotrophs and cyanobacteria are consistent with a cyanobacterial origin of plastids.

At4g33680, the locus encoding LL-DAP-AT, was previously identified based on the phenotype of a point mutant that caused aberrant growth defects and cell death named agd2 (Song et al., 2004). Based on their finding that the AGD2 protein was able to transaminate Lys with a physiologically implausible K_m of 58.8 mM, Song et al. (2004) proposed that it might be involved in Lys metabolism. In fact, as reported here the K_m for LL-DAP is 830-fold lower than the value for Lys. In addition, a 1,000-fold excess addition of Lys to an assay did not inhibit LL-DAP-AT activity (data not shown). Thus, Lys itself is not a substrate for nor does it inhibit LL-DAP-AT. In seeking a plausible explanation for these disparate observations, it did seem possible that with the very high concentrations of Lys (100 mM) used by Song et al. (2004) that even small amounts of contaminants might have given rise to their results. Since commercially available Lys is prepared by bacterial fermentation, it was not surprising to find that analysis of our own Lys stock revealed the presence of detectable amounts of LL-DAP and m-DAP.

The data reported here indicate that At4g33680 encodes an LL-DAP-AT that can function in Lys synthesis. Whether it is the only enzyme that can convert THDPA to LL-DAP in Arabidopsis is not absolutely known. Although its closest paralog in Arabidopsis, encoded by At2g13810, did not show LL-DAP-AT activity when expressed in E. coli, it is important to mention that this negative evidence does not rule out the possibility that it has this activity. However, a T-DNA-insertional, knockout allele of At4g33680 has been found to be embryo lethal, indicating that this gene is essential (Song et al., 2004). Further analysis will be necessary to resolve the question of whether At4g33680 is a unique gene or is a member of a functionally redundant gene family.

The initial identification of LL-DAP-AT was made by measuring the conversion of LL-DAP to THDPA, a reaction that runs in the reverse direction relative to Lys synthesis. The activity of the enzyme proved to be highly specific in that it was able to distinguish between DAP isomers and several acceptors commonly used by aminotransferases. The LL-DAP-AT was unable to use m-DAP, an isomer of LL-DAP. In addition, 2-OG was used as amino acceptor specifically over pyruvate and oxaloacetate. LL-DAP-AT also proved to be capable of the physiologically significant forward activity with an initial rate that is disfavored by 50-fold compared with the reverse activity. Despite this unfavorable feature, the enzyme was demonstrated to function in the forward direction under physiological conditions by the fact that it is able to substitute for the lack of succinyltransferase and deacylase activities in the dapD and dapE mutants of E. coli. Barring the possibility that the molecular construction used to produce the recombinant enzyme negatively affected its catalytic properties, it is very likely that the physiological concentrations of substrates offset the unfavorable V_max ratio. The level of Glu in the chloroplast stroma has been reported for several plant species to be in the range of 14 to 73.6 mM (Winter et al., 1993, 1994; Leidreiter et al., 1995), well above the 2.0 mM K_m[Glu] of the LL-DAP-AT. Although 2-OG concentration has been less well documented, it was found to be 70 µM in the chloroplast stroma of spinach (Spinacia oleracea) and has been estimated, based on the properties of a 2-OG/malate transporter in Arabidopsis, to be in the low micromolar range (Weber and Flugge, 2002). Such a concentration is well below the 8.3 mM K_m[2-OG] of the LL-DAP-AT. The E. coli cytoplasm also contains a high Glu/2-OG ratio (Cayley et al., 1991), which, just as in the chloroplast stroma, drives biosynthetic transaminations, and explains why LL-DAP-AT can replace DapD and DapE in E. coli. A further indication that the conditions in plastids favor the forward reaction for LL-DAP-AT comes from measurement of the activities of enzymes acting before and after the LL-DAP-AT. In extracts prepared from maize (Zea mays) embryo cultures, the LL-DAP-AT forward activity (estimated based on the ratio of forward and reverse activity of the pure Arabidopsis recombinant enzyme) was about 0.16 nmol min^–1 mg^–1 protein. Extracts from the same culture showed DapA activity of 1.3 nmol min^–1 mg^–1 protein, DapB activity of 17.0, DapF activity of 29.0, and LysA activity of 48.0 (Chatterjee et al., 1994). All of these activities are an order of magnitude or more above the activity of the LL-DAP-AT. Although the concentrations of THDPA and LL-DAP have never been directly measured in plants, based on the enzyme-specific activities it is likely that the THDPA concentration would be higher than that of LL-DAP. Another interesting observation that can be gleaned from the analysis of Lys biosynthesis enzyme activities from maize embryo cultures is that the overall rate of Lys synthesis was 0.53 nmol min^–1 mg^–1 protein (Hudson et al., 2005), in the same range as the forward rate of LL-DAP-AT (calculated from the OAB activity in Table I, based on the ratio of forward and reverse activity of the pure enzyme in Table III). Thus, it appears that in the case of the maize embryo culture, LL-DAP-AT may limit the biosynthesis of Lys under some conditions. It is recognized that the first enzyme of the pathway, dihydrodipicolinate synthase, is feedback regulated by Lys and plays a primary role in regulating the pathway (Shaul and Galili, 1993). LL-DAP-AT activity may play a role in limiting the pathway when dihydrodipicolinate synthase is not inhibited by Lys.

The discovery of LL-DAP-AT and the hint that it may be a factor limiting the rate of Lys biosynthesis could have implications for agriculture. Animals cannot produce Lys and so they rely on a dietary source, which is derived primarily from crop plants. Since some crops do not accumulate enough Lys to allow them to be used as complete nutritional sources, there has been significant interest in improving nutritional quality by enhancing Lys content (Mazur et al., 1999). It is known that in plants the control of Lys homeostasis is complex with degradation playing as significant a role as biosynthesis (Galili et al., 2001; Zhu and Galili, 2004). Therefore, the discovery of LL-DAP-AT has completed our understanding of the exact pathway by which plants synthesize Lys and has revealed another potential target for plant improvement.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Microbial Strains

The microbial strains used in this study are listed along with their contributors: Escherichia coli strains AT980, AT984, and AT999 (Coli Genetic Stock Center), JC7623 (Cranenburgh et al., 2001), BL21(DE3)/pET28-CgDDH expressing Corynebacterium glutamicum Ddh (D.I. Roper, University of Warwick), Synechocystis sp. PCC6803 and Bacteriophage P1kc (American Type Culture Collection, nos. 27184 and 25404-B1, respectively), Synechococcus sp. PCC7942 (B. Zilinskas, Rutgers University), Bacillus subtilis 168 (Bacillus Genetic Stock Center), Rhizobium tropici USDA9030 (U.S. Department of Agriculture-Agricultural Research Service National Rhizobium Germplasm Collection), and Agrobacterium tumefaciens GV3101 (Koncz and Schell, 1986). Molecular biology techniques were performed as generally described by Sambrook et al. (1989). E. coli strain AOH1 was constructed by transduction of dapD::Kan2 from JC7623 into AT984 using P1kc. Replacement of dapD⁺ with dapD::Kan2 was confirmed by PCR using primers 5'-AATGGAGATCGGCCAGAAAAA-3' and 5'-GGTGCCCGAATTACAACCATT-3'.

Plants and Growth Conditions

Arabidopsis (Arabidopsis thaliana) Col7 (Arabidopsis Biological Resource Center), Glycine max, spinach (Spinacia oleracea), Brassica napus, and pea (Pisum sativum) Progress 9 were grown in peat-based PRO-MIX BX and fertilized with Peter's nutrients 20:20:20 (N:P:K) in a growth chamber with 16-h-light and 8-h-dark periods. The temperature was 24°C during the light period and 20°C during the dark. Light intensity was 120 µE m^–2 s^–1. Arabidopsis was also grown axenically in Murashige and Skoog liquid medium with minimal organics (Sigma-Aldrich product no. M6899). Surface-sterilized seed were sown into 50-mL medium in a 250-mL Erlenmeyer flask and were grown for 10 d with constant mixing on an orbital shaker at 50 rpm. Convallaria majalis was collected from the field. Maize (Zea mays) was from an embryogenic culture (Singh et al., 1988). Chlamydomonas reinhardtii and Physcomitrella patens were grown as described (Gorman and Levine, 1966; Schaefer et al., 1991).

cDNA Cloning and Protein Expression

The cDNA derived from At4g33680 was amplified by reverse transcription (RT)-PCR using the primers 5'-GGGGCATTGGAAGGAGATATAACCATGGCAGTCAATACTTGCAAATGT-3' and 5'-GGGGGTCGACTCATTTGTAAAGCTGCTTGAATCTTCG-3'. Total RNA was isolated from 25-d-old Arabidopsis leaf using Trizol reagent (Life Technologies). RT was carried out with Superscript II RNAse H– Reverse Transcriptase system (Invitrogen, catalog no. 18064-014) using 1 µg of total RNA and an oligo(dT) primer. PCR was then carried out with the gene-specific primers using 12 pM of each primer, 1 mM MgSO₄, 0.5 mM of each of the four deoxynucleotide triphosphates, 2 µL RT reaction, and 1 unit of Platinum Pfx DNA polymerase using the following conditions: 1 cycle at 94°C, 2 min; and 36 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 2 min. The DNA fragment was digested with NcoI and SalI and cloned into pET30b to produce pET30-AtDAT. The recombinant protein lacks the first 39 amino acids of the At4g33680 protein and carries hexa-His and S-TAG sequence derived from pET30b at its amino terminus. Synechocystis sp. sll0480 was amplified from genomic DNA by PCR using the primers 5'-GGGGGGATCCATGGCCAGTATCAACGACAAC-3' and 5'-GGGGGTCGACCTAACCCAATTTGAGGGTGGA-3'. The DNA fragment was digested with BamHI and SalI and cloned into pET30b to produce pET30-SsDAT. The recombinant protein derived from this plasmid carries the affinity tags fused to the amino terminus of the full-length sll0480 protein. pET30b-AtDAT and pET30b-SsDAT were transformed into E. coli BL21-CodonPlus-RIPL. Plasmids for functional complementation of E. coli dap mutants were produced by subcloning the XbaI and SalI fragment from pET30-AtDAT or pET30-SsDAT into pBAD33 (Guzman et al., 1995) to produce pBAD33-AtDAT and pBAD33-SsDAT. The fusion proteins produced from the pBAD33 constructs were identical to those from the pET30b constructs.

For protein expression and purification, the strains were grown on Luria-Bertani (LB) medium at 37°C to an OD600 nm of 0.5 and protein expression was then induced with 1 mM isopropylthio--galactoside for 4 h at 25°C. Cells were lysed by sonication in a solution of 50 mM sodium phosphate and 300 mM NaCl (pH 8.0). The soluble fraction was incubated with Talon metal affinity agarose (CLONTECH no. 8901-2), washed three times with lysis buffer containing 10 mM imidazole, and eluted with 300 mM imidazole. The protein was concentrated in an Amicon Ultra 30,000 molecular weight cutoff (MWCO) ultrafilter, replacing the elution buffer with 100 mM HEPESKOH, pH 7.6. For the E. coli culture expressing C. glutamicum Ddh, the protein was not purified because it comprised approximately 90% of the soluble protein. The preparation converted m-DAP to THDPA at a rate of 14 µmol min^–1 mg^–1 protein at 30°C.

Functional Complementation and Enzyme Assays

In functional complementation dap mutant strains were transformed with either the plasmid vector or with LL-DAP-AT expression plasmids. Transformants were selected on LB medium supplemented with 50 µg mL^–1 DAP (DL-,-DAP, Sigma-Aldrich product no. D-1377) and 34 µg mL^–1 chloramphenicol. Individual colonies were then replica plated onto NZY medium (5 g L^–1 NaCl, 2 g L^–1 MgSO₄-7H₂O, 10 g L^–1 caseine hydrolysate, 5 g L^–1 yeast extract, 15 g L^–1 agar) supplemented with 0.2% (w/v) Ara without or with 50 µg mL^–1 DAP. The cultures were grown at 30°C for 48 h.

For enzyme assays of crude proteins, extracts were prepared by grinding tissue in liquid nitrogen with 100 mM HEPESKOH (pH 7.6), followed by centrifugation at 10,000g for 15 min, and then buffer exchange using an Amicon Ultra 30,000 MWCO filter. The OAB assay contained in 1 mL 100 µmol HEPESKOH (pH 7.6), 0.5 µmol amino donor, 2.0 µmol 2-OG, and 1.25 mg OAB, and crude soluble protein or pure protein. Reactions were incubated at 30°C and the A (440 nm) measured continuously. Quantitative assay of the physiologically reverse activity was measured in 1 mL containing 100 µmol HEPESKOH (pH 7.5), 0.3 µmol NADPH, 50 µmol NH₄Cl, 0.5 µmol LL-DAP, 5 µmol 2-OG, 16 µg CtDdh, and pure LL-DAP-AT. The reaction was incubated at 30°C, and the decrease in A₃₄₀ was measured. Quantitative assay of the physiologically forward reaction was measured in 1 mL containing 100 µmol HEPESKOH (pH 7.5), 0.5 µmol NADP⁺, varying concentrations of m-DAP, 0.3 µmol thio-NAD⁺, 0.3 µmol CoA, 0.5 µmol Glu, and 32 µg Ddh. The reaction was run to completion, determined by monitoring the increase in A₃₄₀. Then 200 µg of 2-OG dehydrogenase (0.625 µmol min^–1 mg^–1 protein) and pure LL-DAP-AT were added. The kinetic constants were calculated by nonlinear regression analysis using GraphPad Prizm version 4.03.

	ACKNOWLEDGMENTS

 
The authors wish to acknowledge Jin Gu Gang for outstanding technical assistance.

Received October 6, 2005; returned for revision November 2, 2005; accepted November 3, 2005.

	FOOTNOTES

 
¹ This work was funded by a grant from the National Science Foundation (IBN–0449542 to T.L. and C.G.) and the National Institutes of Health Predoctoral Fellowship (GM069264 to A.O.H.) and Institutional Training Grant (GM55145).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Thomas Leustek (leustek{at}rutgers.edu).

^[W] The online version of this article contains Web-only data.

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	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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