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

In Escherichia coli the first common precursor of all tetrapyrroles, 5-aminolevulinic acid, is synthesized from glutamyl-tRNA (Glu-tRNAGlu) 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-tRNAGlu 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.

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 GluTR⅐GSA-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 GluTR⅐GSA-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 GluTR⅐GSA-AM complex.
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 cocrystallization of the competitive inhibitor glutamycin representing the 3Ј-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. Structurebased 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 * 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.
ʈ To whom correspondence should be addressed. Tel.: 49-531-391-5808; Fax: 49-531-391-5854; E-mail: j.moser@tu-bs.de. 1 The abbreviations used are: ALA, 5-aminolevulinic acid; DTT, 1,4dithio-D,L-threitol; GluRS, glutamyl-tRNA synthetase; GluTR, glutamyl-tRNA reductase; Glu-tRNA Glu  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 GluTR⅐GSA-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.

EXPERIMENTAL PROCEDURES
Overexpression and Purification of E. coli GluTR-Details for recombinant production, refolding, and purification of recombinant E. coli GluTR have been published elsewhere (7).
Construction of the Gene for the GluTR Mutant A338Stop by Sitedirected 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: 5Ј-GCGTG-GCTGCGATAACAAAGCGCCAGCGAAAC-3Ј (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 Ϯ 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 g/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 Ͻ5% 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 g/ml ampicillin to an A 578 nm of 0.6. After the addition of another 100 g/ml ampicillin and 400 M 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 ϫ 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 (ϳ200 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 (Vivascience, 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 ϳ50 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 ϫ g at 4°C. From the supernatant 300 l were incubated with 2.5 l 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 l 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 ϫ 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 ϫ g. The samples were eluted into 20 l 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   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). 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 antirabbit 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 M wild type GluTR or GluTR deletion mutant A338Stop, respectively, and 1 M of GSA-AM were analyzed in 50 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 10 mM MgCl 2 , 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 M L-glutamate, 4 mM ATP, 2 mM NADPH, 500 M PLP, 1 M E. coli glutamyl-tRNA synthetase (GluRS), and 20 M E. coli tRNA preparation containing ϳ37% tRNA Glu acceptor activity prepared as described elsewhere (14). 100 l of this assay mixture were incubated for 10 min at 4°C or alternatively for 2 min at 37°C. After dilution with 300 l of assay buffer, 1 l 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. Coprecipitation and immunodetection was then performed analogous to the experiments using cell-free extracts as described above.
Analysis of GluTR⅐GSA-AM Interaction by Gel Filtration according to Hummel and Dreyer-The Hummel and Dreyer method (15)  Reaction products were analyzed via HPLC on a Waters Bondapack TM C 18 reversed phase column (3.9 ϫ 150 mm, 125 Å pore size, 10 m 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 M 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, and coordinates for the Synechococcus GSA-AM (10) were obtained from the ID code 2GSA. Sequence align-ments 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).

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 GluTR⅐GSA-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 GluTR⅐GSA-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 GluTR⅐GSA-AM complex detectable via co-immunoprecipitation is present in E. coli cell-free extracts.
Complex Formation between Purified E. coli GluTR and GSA-AM Is Glutamyl-tRNA-and Cofactor-independent-To further study the prerequisites for the observed interaction between E. coli GluTR and GSA-AM, co-immunoprecipitation experiments using recombinant purified enzymes at protein concentrations of 1 M were performed. Incubation of the assay mixture prior to immunoprecipitation was carried out in the presence and absence of Glu-tRNA Glu and catalytically important cofactors such as NADPH and PLP at both 4°C and 37°C. At both preincubation temperatures the GluTR⅐GSA-AM complex was precipitated from the assay mixture independently of the addition of the substrate Glu-tRNA Glu and the NADPH and PLP cofactors. Identical results were obtained by using either anti-GluTR or anti-GSA-AM antibodies for the precipitation. The data are shown for anti-GSA-AM antibodies in Fig. 3A, lane 3. The pre-immune serum control is shown in Fig. 3A, lane 1. From these results we conclude that complex formation at the employed protein concentrations is not dependent on the flow of metabolites through the GluTR⅐GSA-AM complex.
GluTR Dimerization Enhances GluTR⅐GSA-AM Complex Formation-To study the role of the C-terminal domain in complex formation in vitro co-immunoprecipitation experiments were performed using the GluTR-A338Stop mutant lacking the dimerization domain. Gel filtration chromatography indicated a native relative molecular mass of 39,000 Ϯ 3,000 Da for the truncated protein. Based on these results it was concluded that the GluTR-A338Stop-mutant (40,112 Da calculated molecular mass) is a globular monomeric two-do-main GluTR variant. The function of the 80 C-terminal residues (representing 19% of the overall GluTR sequence) is the formation of the dimerization domain responsible for the Vshaped quaternary structure of the wild type GluTR. An intact dimerization domain is also important for the enzymatic activity of the E. coli GluTR as indicated by only 5% residual activity of the mutant enzyme compared with the wild type enzyme. The monomeric enzyme was tested for complex formation at a concentration of 1 M in the presence or absence of Glu-tRNA Glu and associated cofactors. In comparison with the dimeric wild type enzyme the amount of precipitated enzyme was significantly reduced, but there was still a detectable interaction (Fig. 3B). The amount of wild type GluTR co-immunoprecipitated in the GluTR⅐GSA-AM complex was ϳ50% of GluTR precipitated using anti-GluTR antibodies (Fig. 3A, compare  lanes 2 and 3). However, only about 20% of complex bound GluTR-A338Stop was detected compared with freely precipitated GluTR-A338Stop (Fig. 3B, compare lanes 2 and 3). These experiments suggested an important role of the C-terminal dimerization domain in the facilitation of complex formation. From these data we conclude that the dimeric V-shaped GluTR structure is an important prerequisite for the interaction of both enzymes. Nevertheless, complex formation was not solely dependent on those C-terminal residues but also required the residual GluTR molecule as indicated by the small amount of observed co-precipitate using GluTR-A338Stop.
Gel Filtration Analysis of the GluTR⅐GSA-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 M GSA-AM until a stable absorption at 280 nm was observed. Subsequently, a sample containing 10 M GluTR in addition to the 10 M 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 M. 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 M bovine serum Toward a Function of the GluTR⅐GSA-AM Complex by Way of Sequential Versus Coupled ALA Formation-To identify a function for the GluTR⅐GSA-AM complex, the enzymatic conversion of [ 14 C]Glu-tRNA Glu into ALA via the highly reactive GSA intermediate was compared for the two consecutive enzymatic reactions and the coupled reactions. Resulting reaction products were identified and quantified by scintillation counting after reversed phase HPLC chromatographic separation. In vitro reactions of GluTR alone with the substrate [ 14 C]Glu-tRNA Glu led to the formation of [ 14 C]GSA, with a retention time of 7.5 min and, because of spontaneous substrate hydrolysis, to a [ 14 C]Glu peak at 5 min. Besides those two well characterized products a third 14 C-labeled compound, with a retention time of 2.6 min, was reproducibly detected as the result of GluTR catalysis (Fig. 5A). When GSA-AM was added to this product mixture only [ 14 C]GSA was converted into [ 14 C]ALA. The additional compound at 2.6 min was no substrate for GSA-AM catalysis (Fig. 5B). In coupled in vitro assays, allowing complex formation prior to substrate addition, this additional compound was not detectable (Fig. 5C). On the basis of these observations one might speculate that one essential role of GluTR⅐GSA-AM complex formation is to prevent the side reaction of the reactive GSA aldehyde species, possibly with cellular compounds or the solvent. Another possible reaction has been described earlier during the chemical synthesis of GSA in which a cyclization of GSA to 2-hydroxy-3-aminotetrahydropyran-1-one was described (23). To date no physical characterization of that compound has been reported. Because of the minimal amounts of intermediate formed in the assay mixture (Ϸ5 pmol) no further characterization of this side product was possible. The experiments clearly demonstrated that the semialdehyde species was protected from an inefficient side reaction by the presence of GSA-AM. However they do not rule out the possibility that a very rapid GSA-AM reaction in the coupled assay might also result in the protection of GSA. Nevertheless, these findings are in clear agreement with the postulated substrate channeling pathway as indicated by x-ray crystallography and by modeling experiments (5) in which the intermediate aldehyde is prevented from exposure to the aqueous environment.
The current investigation was one of the rare cases in which the structural biology of related enzymes from different organisms directly give the answer to a metabolic question. The present investigation demonstrates the existence of a GluTR⅐GSA-AM complex in E. coli, which might indicate that the original structure-based complex model can be regarded of general significance for the GluTR⅐GSA-AM interaction in plants, archaea, and all bacteria synthesizing ALA from Glu-tRNA Glu .