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J. Biol. Chem., Vol. 280, Issue 19, 18568-18572, May 13, 2005

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Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis^*

Corinna Lüer, Stefan Schauer, Kalle Möbius, Jörg Schulze¶, Wolf-Dieter Schubert¶, Dirk W. Heinz¶, Dieter Jahn, and Jürgen Moser||

From the Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany, Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Schafmattstrasse 20, CH-8093 Zürich, Switzerland, and ^¶Division of Structural Biology, German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Received for publication, January 13, 2005 , and in revised form, March 4, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
In Escherichia coli the first common precursor of all tetrapyrroles, 5-aminolevulinic acid, is synthesized from glutamyl-tRNA (Glu-tRNA^Glu) in a two-step reaction catalyzed by glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM). To protect the highly reactive reaction intermediate glutamate-1-semialdehyde (GSA), a tight complex between these two enzymes was proposed based on their solved crystal structures. The existence of this hypothetical complex was verified by two independent biochemical techniques. Co-immunoprecipitation experiments using antibodies directed against E. coli GluTR and GSA-AM demonstrated the physical interaction of both enzymes in E. coli cell-free extracts and between the recombinant purified enzymes. Additionally, the formation of a GluTR·GSA-AM complex was identified by gel permeation chromatography. Complex formation was found independent of Glu-tRNA^Glu and cofactors. The analysis of a GluTR mutant truncated in the 80-amino acid C-terminal dimerization domain (GluTR-A338Stop) revealed the importance of GluTR dimerization for complex formation. The in silico model of the E. coli GluTR·GSA-AM complex suggested direct metabolic channeling between both enzymes to protect the reactive aldehyde species GSA. In accordance with this proposal, side product formation catalyzed by GluTR was observed via high performance liquid chromatography analysis in the absence of the GluTR·GSA-AM complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
In plants, green algae, archaea, and most bacteria the common precursor molecule of all tetrapyrroles, 5-aminolevulinic acid (ALA),¹ is synthesized from tRNA-bound glutamate (Glu-tRNA^Glu) in a two-step reaction (1, 2). In the first step, the NADPH-dependent glutamyl-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA). In a subsequent transamination reaction this aldehyde is transformed into ALA by the pyridoxal 5'-phosphate-dependent enzyme glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM) (3).

Recently the catalytic mechanism of GluTR and its structural basis have been elucidated in a combined biochemical and structural investigation using the recombinant enzyme from the extreme thermophilic archaean Methanopyrus kandleri (4, 5). The crystal structure of GluTR reveals an unusual extended V-shaped dimer with each monomer consisting of three distinct domains arranged along a curved "spinal" -helix. The N-terminal catalytic domain specifically recognizes the glutamate moiety of the substrate. The active site was identified by co-crystallization of the competitive inhibitor glutamycin representing the 3'-terminal end of the natural substrate. During catalysis a nucleophilic cysteine residue attacks the aminoacyl linkage of the glutamate to its cognate tRNA and generates an enzyme-bound thioester intermediate. This intermediate has been biochemically trapped and visualized. For this purpose a new purification strategy for the Escherichia coli enzyme has been developed (6, 7). The thioester intermediate gets finally reduced by direct hydride transfer from NADPH to form GSA and to release tRNA^Glu. The nucleotide cofactor is supplied by the second distinct domain, the NADPH binding domain. An additional C-terminal domain of GluTR is responsible for the dimerization of the unusual V-shaped molecule. Structure-based alignments of amino acid sequences from different sources have revealed a high degree of sequence identity (8). These findings along with the biochemical data for the E. coli enzyme indicate that the M. kandleri enzyme can be regarded as a model system representing all GluTR enzymes.

ALA synthesis requires the concerted action of the two enzymes GluTR and GSA-AM that are metabolically linked by the highly reactive aldehyde GSA. A half-life of less than 4 min was determined for GSA at physiological pH in aqueous solution (9). Based on the three-dimensional structures of GluTR from M. kandleri and GSA-AM from Synechococcus sp. (10) a hypothetical model ensuring efficient ALA synthesis was proposed (5). We realized that the open space delimited by the GluTR monomers is remarkably similar to the volume occupied by GSA-AM. In silico the dimeric GSA-AM was placed into the open space of the V-shaped GluTR-dimer. Both enzymes were docked along their 2-fold symmetry axes leading to a model complex with a high degree of surface complementarity (in Fig. 1 the analogous E. coli model complex is shown). Independently, tRNA^Glu was docked in a single plausible position on the GluTR protein. The resulting combined model of the ternary complex (GluTR, tRNA, and GSA-AM) did not lead to steric clashes. Additional strong evidence for the model complex came from the observation that the putative active site entrance of each GSA-AM monomer is positioned opposite a partly opened depression of the catalytic domain of GluTR. This depression and the GluTR active site pocket are separated from each other only by the conserved arginine 50 (M. kandleri GluTR numbering). In our current hypothesis the GluTR product GSA leaves the enzyme via this "back door" of the GluTR active site pocket and subsequently enters the active site of GSA-AM. This way direct channeling of labile GSA to the active site of GSA-AM without exposure to the aqueous environment is possible. Here we provide the first experimental evidence for the GluTR·GSA-AM complex using two independent biochemical techniques. Additionally, we created the homologous E. coli model complex in an in silico experiment (Fig. 1) supporting our results.

View larger version (79K): [in this window] [in a new window]   FIG. 1. Model complex of glutamyl-tRNA reductase (dark gray) and glutamate-1-semialdehyde 1,2-aminomutase (light gray) from E. coli. The docking model was generated based on the x-ray structures of GluTR from M. kandleri (10) and GSA-AM from Synechococcus (15).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Construction of the Gene for the GluTR Mutant A338Stop by Site-directed Mutagenesis—A deletion mutant lacking the dimerization domain (GluTR-A338Stop) was generated using the plasmid pBKCwt (6) and the QuikChange^TM kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The following oligonucleotide was employed to introduce a stop codon into the E. coli GluTR sequence: 5'-GCGTGGCTGCGATAACAAAGCGCCAGCGAAAC-3' (stop-codon underlined).

Purification and Characterization of the E. coli GluTR Deletion Mutant A338Stop—Purification and refolding of the truncated protein was performed in analogy to the wild type enzyme (7). The yield of refolded protein was 4 mg from 0.6 g of inclusion bodies. In the final concentrated fraction a single protein band on a SDS-polyacrylamide gel was visible after Coomassie Blue staining. The calculated molecular mass of the mutant enzyme deduced from the gene sequence (40,112 Da) was experimentally confirmed using electrospray ionization mass spectrometry (4) to be 40,110 ± 5 Da (data not shown). Edman degradation revealed an identical N-terminal amino acid sequence (first 15 amino acids) as derived from the cloned gene sequence. N-terminal protein sequencing was performed using an Applied Biosystems 454 sequencer (Applied Biosystems). To provide further evidence for proper refolding of GluTR-A338Stop circular dichroism spectroscopy was employed. CD spectroscopy was carried out on a Jasco J-810 spectropolarimeter (Jasco, Gross-Umstadt, Germany). Protein solutions of GluTR and GluTR-A338Stop were dialyzed against 20 mM Tris-HCl, pH 8.0, containing 10 mM NaCl. The concentration of the protein solution was 500 µg/ml. In quartz cuvettes of 1-mm path length, CD spectra over a range of 190–250 nm were recorded at room temperature as an average of 5 scans. Thermal unfolding was carried out in the range of 20–100 °C at a rate of 1 °C/min. The molar ellipticity was measured at 220 nm every 2 °C. Samples were allowed to equilibrate for at least 20 min at 20 °C before starting each scan. Both GluTRwt and GluTR-A338Stop were used for the following measurements. Using CD spectroscopy in the far-UV spectral region, we detected no significant differences compared with the wild type enzyme. Thermal unfolding experiments followed by CD spectroscopy indicated a comparable temperature range (80–85 °C) for the unfolding of the mutant protein (data not shown). These data demonstrate that the C-terminal domain of GluTR comprising residues 338–418 is not essential for the proper folding of the catalytic and the NADPH-binding domain. For the GluTR-A338Stop mutant a residual activity of <5% compared with wild type enzyme was detected using the standard GluTR depletion assay (4).

Large Scale Overproduction, Purification, and Characterization of E. coli GSA-AM—E. coli BL21 (DE3) carrying pLIpopC (11) was cultivated in 1 liter of LB medium containing 100 µg/ml ampicillin to an A_{578 nm} of 0.6. After the addition of another 100 µg/ml ampicillin and 400 µM isopropyl--D-thiogalactopyranosid, cells were grown for an additional 3 h and harvested by centrifugation. The bacterial cell pellet (4 g) was resuspended in 20 ml of 100 mM PIPES-NaOH, pH 6.8, containing 5 mM DTT, 1 mM EDTA (buffer A). Cells were disrupted by sonication, and cell debris was removed by centrifugation for 45 min at 50,000 x g at 4 °C. The supernatant was loaded onto a 25-ml DEAE-Sepharose Fast Flow column (XK 16 column, Amersham Biosciences, Freiburg, Germany) equilibrated with buffer A. After washing the column with 2 column volumes of buffer A, proteins were eluted with a linear gradient of 5 column volumes ranging from 0 to 1 M NaCl in buffer A. Fractions containing GSA-AM were pooled (200 mg of total protein) and dialyzed against 20 mM HEPES-NaOH, pH 7.9, 10 mM NaCl, 5 mM DTT (buffer B) at 4 °C. This solution (40 ml) was subsequently loaded on a MonoQ HR 10/10 column (Amersham Biosciences) equilibrated with buffer B. The column was washed with 2 column volumes of buffer B. Bound proteins were eluted using a linear gradient (150 ml) with a concentration of 10 mM to 1 M NaCl in buffer B. GSA-AM-containing fractions were pooled and concentrated by ultrafiltration using a Vivaspin-15 centrifugal concentrator with a molecular weight cut-off of 10,000 (Viva-science, Hannover, Germany). A final volume of 2 ml with a protein concentration of 45 mg/ml was chromatographed on a Superdex 75 prep grade, high load 26/60 gel filtration column (Amersham Biosciences) equilibrated previously with 20 mM HEPES-NaOH, pH 7.9, 100 mM NaCl, 10 mM DTT at a flow rate of 2.0 ml/min. Fractions containing GSA-AM were pooled and concentrated to 30 mg/ml (Vivaspin-15 concentrator, molecular weight cut-off 30,000). The newly established purification procedure for the E. coli GSA-AM yielded 50 mg of protein/liter of bacterial culture purified to apparent homogeneity as judged by SDS-PAGE. The integrity of the enzyme preparation was experimentally verified by electrospray ionization mass spectrometry and by N-terminal protein sequencing as described above (data not shown). Analytical gel filtration chromatography, performed as described previously (12) resulted in a single, well resolved peak. The cofactor absorption spectrum indicated a peak at 330 nm and another peak at 430 nm as described previously (3). The CD spectrum of the E. coli GSA-AM was comparable with that of the Synechococcus enzyme (10).

Co-immunoprecipitation Experiments Using Cell-free E. coli Extracts—Polyclonal rabbit antibodies against recombinant E. coli GluTR and GSA-AM were generated by Eurogentec (Seraing, Belgium). For co-immunoprecipitation using cell free extracts, a total of 0.5 g of aerobically grown E. coli BL21 (DE3) cells were harvested in the early exponential phase. The bacterial cell pellet was resuspended in 5 ml of Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 10 mM DTT, and 0.5% (v/v) of the detergent Nonidet P-40 (lysis-buffer). Cells were disrupted by sonication, and cell debris was removed by centrifugation for 30 min at 40,000 x g at 4 °C. From the supernatant 300 µl were incubated with 2.5 µl of anti-GluTR (5 mg/ml) or anti GSA-AM serum (8 mg/ml), respectively, by gentle shaking for 90 min at 4 °C. After the addition of 15 µl of a 1:1 slurry of protein A-Sepharose CL-4B (Amersham Biosciences) in lysis buffer, the mixture was further incubated for 90 min at 4 °C. The immunoadsorbent was recovered by centrifugation for 5 min at 500 x g and washed three times by resuspension in Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 1 mM EDTA, and 0.5% (v/v) Nonidet P-40 and centrifugation for 5 min at 500 x g. The samples were eluted into 20 µl of SDS loading buffer (Sigma).

Immunoblot Analysis—Protein samples eluted from protein A-Sepharose were heated for 5 min at 99 °C and subjected to 9% SDS-PAGE using standard techniques (13). The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes using a Trans-Blot apparatus (semi-dry transfer cell, Bio-Rad) according to the manufacturer's instructions. The membrane was first incubated with anti-GSA-AM or anti-GluTR rabbit antibodies (1:30,000 in phosphate-buffered saline (13) with 3% bovine serum albumin), washed three times with phosphate-buffered saline, and then incubated with alkaline phosphatase-conjugated sheep anti-rabbit antibodies (1:20,000 in phosphate-buffered saline with 3% bovine serum albumin) from Pierce (Bonn, Germany). The detection of immunoreactive bands was performed using the nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate color developing method from Promega (Mannheim, Germany).

Co-immunoprecipitation Experiments Using Purified Recombinant E. coli GluTR and GSA-AM—For co-immunoprecipitation using purified enzymes 1 µM wild type GluTR or GluTR deletion mutant A338Stop, respectively, and 1 µM of GSA-AM were analyzed in 50 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 5 mM DTT, 10% (v/v) glycerol, 0.1% (w/v) bovine serum albumin, and 0.05% (v/v) Tween 20 (assay buffer) containing 500 µM L-glutamate, 4 mM ATP, 2 mM NADPH, 500 µM PLP, 1 µM E. coli glutamyl-tRNA synthetase (GluRS), and 20 µM E. coli tRNA preparation containing 37% tRNA^Glu acceptor activity prepared as described elsewhere (14). 100 µl of this assay mixture were incubated for 10 min at 4 °C or alternatively for 2 min at 37 °C. After dilution with 300 µl of assay buffer, 1 µl of anti-GluTR (5 mg/ml) or anti-GSA-AM serum (8 mg/ml), respectively, was added to the assay mixture and incubation was continued for 30 min at 4 °C. Co-precipitation and immunodetection was then performed analogous to the experiments using cell-free extracts as described above.

Analysis of GluTR·GSA-AM Interaction by Gel Filtration according to Hummel and Dreyer—The Hummel and Dreyer method (15) is based on the disturbance of the equilibration of a gel filtration column equilibrated with a given concentration of a compound of interest (GSA-AM) by the presence of a weak interaction partner (GluTR). For this purpose a defined amount of the tested interaction partner (GluTR) is injected onto a gel filtration system equilibrated with GSA-AM, and elution profiles are recorded spectroscopically. The binding of GluTR to GSA-AM results in a local deficit of GSA-AM in the eluent, allowing the determination of the binding constant (15, 16). An analytical Superdex 200 PC 3.2/30 (Amersham Biosciences) was used to investigate the binding of GluTR to GSA-AM. The chromatography buffer contained 50 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 5 mM DTT, and 10 µM GSA-AM. After equilibration with 2 column volumes of chromatography buffer a 20-µl injection of various concentrations of GluTR (2–20 µM) dissolved in the chromatography buffer, including 10 µM GSA-AM, was performed. Chromatography was at a flow rate of 100 µl/min. Protein concentration was followed by on-line measuring of absorbance at 280 nm. Analogous experiments were performed using GSA-AM at a concentration of 5 µM.

Enzyme Assays and HPLC Analysis of Reaction Products—The substrate [¹⁴C]Glu-tRNA^Glu was prepared in a bulk reaction using 100 µg of purified E. coli tRNA^Glu (Sigma) and 50 µg of purified E. coli GluRS incubated for 20 min at 37 °C in 30 mM HEPES-NaOH, pH 7.5, containing 15 mM MgCl₂, 25 mM KCl, 3 mM DTT, 4 mM ATP, and 40 µM [¹⁴C]Glu with a specific activity of 284 mCi/mmol (10.5 GBq/mmol). The [¹⁴C]Glu-tRNA^Glu was then separated from the other reaction components as described previously (4). Recombinant E. coli GluRS was purified to apparent homogeneity following published procedures (17).

The GluTR assay mix (50 µl) contained 8 µM GluTR, 6 µM [¹⁴C]Glu-tRNA^Glu in 20 mM HEPES-NaOH, pH 8.1, 1 mM MgCl₂, 30 mM NaCl, 100 µM DTT, 2.5 µM bovine serum albumin, 400 µM NADPH, and 200 µM PLP (standard assay buffer). Reactions were started by the addition of [¹⁴C]Glu-tRNA^Glu and incubated at 37 °C for 8 min. For sequential enzyme analysis the reaction products were formed without the addition of GSA-AM and its cofactor. Formed substances were further incubated with 400 pmol of purified E. coli GSA-AM (8 µM) for 8 min. Reaction products were analyzed via HPLC on a Waters µBondapack^TM C₁₈ reversed phase column (3.9 x 150 mm, 125 Å pore size, 10 µm particle diameter) as described previously (4). The ratio of reaction products was estimated by peak integration.

For coupled enzyme assays the GluTR standard assay described above additionally contained 8 µM E. coli GSA-AM. After incubation for 8 min HPLC analysis revealed the ratio of reaction products. Reactions without GluTR for the different types of enzyme assay served as background controls.

Structure Modeling—The sequences of the E. coli enzymes under study were obtained from the PubMed data base. The coordinates for the M. kandleri GluTR (5) were obtained from the Protein Data Bank data base ID code 1GPJ [PDB] , and coordinates for the Synechococcus GSA-AM (10) were obtained from the ID code 2GSA. Sequence alignments between GluTR from M. kandleri and E. coli, as well for GSA-AM from Synechoccus and E. coli, were carried out using the program ClustalW (18). The modeled E. coli structures were generated using the program BRAGI (19). The model complex was created by placing GSA-AM in the open space delimited by GluTR and docking them along their 2-fold symmetry axes as described previously (5). The image was generated by using PyMOL (20).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
E. coli GluTR and GSA-AM form a Complex in Cell-free Extracts—In silico experiments suggested a complex between GluTR and GSA-AM to protect the labile GSA from hazardous exposure to the aqueous environment. To analyze for the presence of the proposed complex, co-immunoprecipitation experiments were conducted. For this purpose rabbit anti-GluTR and anti-GSA-AM antibodies were generated. The employed strategy involved the recognition of one protein with the specific antibody, immobilization of the antibody-antigen complex on Protein A-Sepharose, and its isolation via centrifugation and washing. In the case of a co-immunoprecipitated interaction partner, this protein was visualized in Western blot experiments using a second antibody directed against it. Co-immunoprecipitation experiments with anti-GluTR and anti-GSA-AM antibodies were performed first with cell-free extracts prepared from wild type E. coli BL21 (DE3) cultures harvested in the early exponential growth phase. These cells contained the natural amounts of both enzymes because none of the corresponding genes was overexpressed. Both of the complementary co-immunoprecipitation experiments resulted in the precipitation of the postulated GluTR·GSA-AM complex. Significant amounts of complexed protein were detected with the corresponding anti-GSA-AM antibody (Fig. 2A, lane 2) and anti-GluTR antibody, respectively (Fig. 2B, lane 2). In the supernatant of the co-precipitates, only residual amounts of the interacting protein partner were detected using Western blot analyses (data not shown). In a control experiment E. coli strain EV61, which carries a disrupted gene for GluTR, was cultivated (21), and a cytosolic extract was prepared analogously to E. coli BL21 (DE3). No immunoprecipitation of GluTR or of GSA-AM by anti-GluTR antibodies was observed (data not shown). In agreement, neither of the pre-immune serums taken prior to the immunization of the rabbits reacted with E. coli GluTR or GSA-AM, respectively (Fig. 2, A and B, lane 1) Because of the known low cellular concentration of both enzymes, only highly concentrated extracts from 20 to 40 mg/ml protein resulted in clear co-immunoprecipitation results. Interestingly, cell-free extracts prepared from stationary phase-grown E. coli did not contain detectable amounts of the GluTR·GSA-AM complex (data not shown). Possibly because of lower heme requirements in the stationary phase, heme-induced GluTR proteolysis decreased cellular GluTR concentrations (22). Clearly, a stable GluTR·GSA-AM complex detectable via co-immunoprecipitation is present in E. coli cell-free extracts.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Western blot analysis of the co-immunoprecipitation of the GluTR·GSA-AM complex from cell-free E. coli extracts. Proteins of an E. coli BL21 (DE3) cell free-extract (40 mg/ml total protein) were immunoprecipitated (IP) using the following antibodies and subsequently Protein A-Sepharose: A, lane 1, with rabbit pre-immune serum (negative control); lane 2, with rabbit anti-GluTR; lane 3, with rabbit anti-GSA-AM; B, lane 1, with rabbit pre-immune serum (negative control); lane 2, with rabbit anti-GSA-AM; lane 3, with rabbit anti-GluTR. Complex formation between E. coli GluTR and GSA-AM was visualized by the detection (DT) of the co-precipitated corresponding protein partner with anti-GSA-AM antibodies in A and anti-GluTR antibodies in B via Western blotting.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Western blot analysis of the co-immunoprecipitation of recombinant purified E. coli GluTR and GSA-AM. Recombinantly produced and purified wild type E. coli GluTR in A and the GluTR-A338Stop variant in B were incubated at a concentration of 1 µM with the same amount of purified E. coli GSA-AM without further addition of substrate or cofactors at 4 °C for 10 min. Immunoprecipitation (IP) was performed with the following antibodies: A and B, lane 1, with rabbit pre-immune serum (negative control); lane 2, rabbit anti-GluTR; lane 3, rabbit anti-GSA-AM. Complex formation between E. coli GluTR and GSA-AM was visualized by Western blot detection (DT) of the co-precipitated corresponding protein partner with anti-GluTR antibodies in A and B. Precipitation of GluTR with anti-GSA-AM antibody compared with the precipitated GluTR with anti-GluTR antibody is lower in the case of the GluTR-A338Stop.

Gel Filtration Analysis of the GluTR·GSA-AM Complex— With the newly established purification procedure for E. coli GSA-AM sufficient quantities of highly pure enzyme were obtained to apply a second independent biochemical method, the Hummel-Dreyer gel filtration chromatography, for complex formation analysis (15). On the basis of our results from the in vitro co-immunoprecipitation experiments we decided to investigate complex formation at room temperature in the absence of substrate and cofactors. A gel filtration column was calibrated for the elution position of GluTR (1.54 ml) and GSA-AM (1.72 ml), respectively. The same gel filtration column was then equilibrated with buffer containing 10 µM GSA-AM until a stable absorption at 280 nm was observed. Subsequently, a sample containing 10 µM GluTR in addition to the 10 µM GSA-AM was injected. The elution profile followed at 280 nm showed in addition to the expected peak for GluTR (1.54 ml) a trough (1.72 ml) in the basal absorbance (Fig. 4). This trough is the result of GSA-AM depletion from the running buffer caused by the binding of GSA-AM to the injected GluTR protein. Analogous chromatographies were conducted with increasing amounts of GluTR ranging from 2 to 20 µM. The size of the trough increased with the concentration of injected GluTR. No troughs were observed when the applied samples did not contain GluTR or when 10–50 µM bovine serum albumin was injected instead. These results clearly indicated the physical interaction of E. coli GluTR and GSA-AM in the absence of substrate and cofactors. From experiments with different protein concentrations an association constant of 10 µM for the complex was deduced.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Gel filtration analysis of the E. coli GluTR·GSA-AM complex. Gel filtration chromatography was performed as outlined under "Experimental Procedures." The arrows indicate the elution volume of GluTR and GSA-AM. A sample of 10 µM GluTR was applied to the gel filtration column equilibrated with chromatography buffer containing 10 µM GSA-AM. Absorption increases at the approximated elution volume of GluTR, whereas a trough is observed at the elution volume of GSA-AM. mAU, milliabsorbance unit.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Comparison of sequential and coupled GluTR- and GSA-AM-mediated ALA formation. The reaction products derived from [14C]Glu-tRNAGlu isolated from different assay mixtures were separated via HPLC chromatography at a flow rate of 0.7 ml/min on the Waters µBondapackTM C18 reversed phase column (3.9 x 150 mm, 125 Å pore size, 10-µm particle diameter). 350-µl fractions were collected, and the [14C] reaction products were quantified using scintillation counting. The C18 reversed phase column was equilibrated using [14C]glutamate (Glu), [14C]ALA, and [14C]GSA (data not shown). The reaction products were identified by scintillation counting. Because of the spontaneous hydrolysis of the substrate Glu-tRNAGlu, an additional peak of [14C]glutamate at 5 min was detected in the chromatograms. A, separation of the products of a sole GluTR reaction with [14C]Glu-tRNAGlu. The additional radioactive compound (2.6 min) besides [14C]GSA (7.5 min) and [14C]Glu (5 min) is marked by an arrow. B, reaction products from A were incubated with GSA-AM for their further conversion into [14C]ALA. Most [14C]GSA (7.5 min) was converted into [14C]ALA; the additional compound of GluTR catalysis marked with an arrow was no substrate for GSA-AM. C, coupled assay with the GluTR·GSA-AM complex and [14C]Glu-tRNAGlu. No additional product peak at a retention time of 2.6 min was detectable. A small amount of the intermediate [14C]GSA was determined besides the [14C]ALA end product.

	FOOTNOTES

 
^* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to D. W. H. and D. J.). 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.

^|| To whom correspondence should be addressed. Tel.: 49-531-391-5808; Fax: 49-531-391-5854; E-mail: j.moser{at}tu-bs.de.

¹ The abbreviations used are: ALA, 5-aminolevulinic acid; DTT, 1,4-dithio-D,L-threitol; GluRS, glutamyl-tRNA synthetase; GluTR, glutamyl-tRNA reductase; Glu-tRNA^Glu, glutamyl-tRNA^Glu; GSA-AM, glutamate-1-semialdehyde 2,1-aminomutase; GSA, glutamate-1-semialdehyde; HPLC, high performance liquid chromatography; PIPES, piperazine-N,N'-bis[2-ethanesulfonic acid]; PLP, pyridoxal 5'-phosphate.

	ACKNOWLEDGMENTS

 
We thank Rita Getzlaff for N-terminal protein sequencing and Manfred Nimtz (both from the German Research Center for Biotechnology, Braunschweig) for mass spectrometry analysis. We are indebted to Nicole Frankenberg-Dinkel for the gel filtration column and Ronja Tasler for advice during the Hummel-Dreyer experiments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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