Two glutamyl-tRNA reductase activities in Escherichia coli

delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.     

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.     

Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies

Glutamyl-tRNA reductase catalyzes the initial step of tetrapyrrole biosynthesis in plants and prokaryotes. Recombinant Escherichia coli glutamyl-tRNA reductase was purified to apparent homogeneity from an overproducing E. coli strain by a two-step procedure yielding 5.6 mg of enzyme per gram of wet cells with a specific activity of 0.47 micromol min(-1)mg(-1). After recombinant production, denatured glutamyl-tRNA reductase from inclusion bodies was renatured by an on-column refolding procedure. Residual protein aggregates were removed using Superdex 200 gel-filtration chromatography. Solubility, specific activity, and long-term storage properties were improved compared to previous protocols. Obtained enzyme amounts of high purity now allow the research on the recognition mechanism of tRNAGlu and high-throughput inhibitor screening.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

Methanopyrus kandleri glutamyl-tRNA reductase.

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Trapping of the substrate-derived acyl enzyme intermediate of purified penicillin-binding protein 1a of Escherichia coli

Purified penicillin-binding protein 1a of Escherichia coli formed an acyl enzyme intermediate with the highly reactive synthetic substrate diacetyl-L-lysyl-D-alanyl-D-lactate at acid pH, although in extremely low yields.     

In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases

Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA(Glu), while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA(Gln), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA(Glu), A. ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNA(Gln) in E. coli in vivo.     

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.     

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-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.     

The glutamyl-tRNA synthetase of Escherichia coli: substrate-induced protection against its thermal inactivation.

The substrates-induced protection against the heat-inactivation of the glutamyl-tRNA synthetase has been investigated. tRNAGlu and ATP protect efficiently the enzyme, whereas glutamate does not. In the presence of tRNAGlu, glutamate induces an additional protection to that given by the tRNAGlu alone. A weak synergism was observed between ATP and tRNAGlu, whereas no synergism was detected between ATP and glutamate. These results suggest that tRNAGlu and ATP, but not glutamate are able to bind to the free enzyme form; glutamate binds only to the Enzyme.tRNAGlu and to the Enzyme.tRNAGlu.ATP complexes. The presence of the three substrates induces a higher stabilization of the enzyme than that expected from the protection observed for the various other substrates combinations, suggesting the existence of a marked synergism between the three substrates against the heat-inactivation of the enzyme. The protection constants determined from this study are similar to the dissociation constants determined by direct binding experiments and to the Km values determined kinetically.     

Purification and properties of Escherichia coli dihydrofolate reductase.

Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.     

Higher specific activity of the Escherichia coli glutamyl-tRNA synthetase purified to homogeneity by a six-hour procedure.

The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells. The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column. The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain. This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli.

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Purification of glutamyl-tRNA reductase from Synechocystis sp. PCC 6803

delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu). The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed. We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme. Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.