The third member of the hemA gene family encoding glutamyl-tRNA reductase is primarily expressed in roots in Hordeum vulgare

Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNA^Gluto glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.     

delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, δ-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA^Glu (tRNA^Glu) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W₁₄ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA^Glu. Other cellular tRNA^Glus present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNA^Glu obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1-semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNA^Glu, and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA^Glu precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena.     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.     

Biosynthesis of the Tetrapyrrole Pigment Precursor, delta-Aminolevulinic Acid, from Glutamate

δ-Aminolevulinic acid (ALA), the common biosynthetic precursor of hemes, chlorophylls, and bilins, is synthesized by two distinct routes. Among phototrophic species, purple nonsulfur bacteria form ALA by condensation of glycine with succinyl-CoA, catalyzed by ALA synthase, in a reaction identical to that occurring in the mitochondria of animals, yeast, and fungi. Most or all other phototrophic species form ALA exclusively from the intact carbon skeleton of glutamic acid in a reaction sequence that begins with activation of the α-carboxyl group of glutamate by an ATP-dependent ligation to tRNA^Glu, catalyzed by glutamyl-tRNA synthetase. Glutamyl-tRNA is the substrate for a pyridine nucleotide-dependent dehydrogenase reaction whose product is glutamate-1-semialdehyde or a similar reduced compound. Glutamate-1-semialdehyde is then transaminated to form ALA. Regulation of ALA formation from glutamate is exerted at the dehydrogenase step through end product feedback inhibition and induction/repression. In some species, end product inhibition of the glutamyl-tRNA synthetase step and developmental regulation of tRNA^Glu level may also occur.     

Light Regulation of delta-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, δ-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA^Glu. ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA^Glu isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity.     

Mechanism of Benzyladenine-Induced Stimulation of the Synthesis of 5-Aminolevulinic Acid in Greening Cucumber Cotyledons: Benzyladenine Increases Levels of Plastid tRNAGlu

The mechanism of the stimulatory effect of a cytokinin, namely,benzyladenine (BA), on the synthesis of 5-aminolevulinic acid(ALA) in cucumber cotyledons was studied. The rate of synthesisof ALA by plastids isolated from BA-treated cotyledons was twicethat by plastids from untreated controls. Western blot analysisof stromal proteins showed that BA did not affect the levelof glutamyl-tRNA synthetase or of glutamate l-semialdehyde (GSA)aminotransferase. Analysis of free amino acids revealed thatBA did not increase the level of glutamate in the stroma. However,the amount of total plastidic RNA was doubled in BA-treatedcotyledons. Northern blot analysis showed that the level ofplastid tRNA^Glu was increased by treatment with BA to the sameextent as that of another plastid tRNA, reflecting an increasein total plastidic RNA. The rate of formation of glutamyl-tRNAwas also doubled in plastids from BA-treated cotyledons. Theresults indicate that stimulation of the synthesis of ALA byBA is due to an increased level of tRNA^Glu in plastids. (Received June 6, 1993; Accepted November 26, 1993)     

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA^Gln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA^Gln and tRNA^Glu with glutamate. This ancient GluRS also separately differentiated to exclude tRNA^Gln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA^Gln and tRNA^Glu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.     

The <Emphasis Type="Italic">Chlamydomonas reinhardtii gtr </Emphasis>Gene Encoding the Tetrapyrrole Biosynthetic Enzyme Glutamyl-tRNA Reductase: Structure of the Gene and Properties of the Expressed Enzyme

Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate by means of a tRNA^Glu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His₆ tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.     

谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成

【背景】大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hem A)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)和血红素合成的影响。【方法】通过Red同源重组敲除与谷氨酸代谢有关的mscS与aroG,构建hemA表达载体并导入基因缺失菌株中。【结果】mscS单敲除或mscS与aroG双敲除对菌体生长无显著影响。与出发菌株相比,单敲除与双敲除菌株的谷氨酸含量均有所增加,ALA含量略微下降,血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后,胞内血红素含量增至47.603μmol/L。【结论】通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成,该结果为增强C5途径的血红素合成提供了新的思路。     

The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System

RNA is an essential component for the enzymic conversion of glutamate to δ-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA^Glu(UUC), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA^Glu(UUC) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA^Glu(UUC) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [³H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA^Glu(UUC) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA^Glu(UUC) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA^Glu(UUC) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [³H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.     

Letter: Nuclear magnetic resonance of protein-nucleic acid interactions. II. The E. coli tRNA-Glu complex with glutamyl-tRNA synthetase

We have observed the 300 MHz high-resolution proton nuclear magnetic resonance spectrum of the Escherichia coli tRNA^Glu complex with the glutamyl-tRNA synthetase. The observations are fitted very well by a computer-simulated spectrum of the E. coli tRNA^Glu itself, with the individual resonances broadened from 45 Hz to 150 Hz because of the increased molecular weight. This indicates that no helical arms open upon complex formation, nor is there evidence for any additional Watson-Crick base-pairs in the complex.     

Isoaccepting mitochondrial glutamyl-tRNA species transcribed from different regions of the mitochondrial genome of Saccharomyces cerevisiae

Mitochondrial glutamyl-tRNA isolated from mitochondria of Saccharomyces cerevisiae was separated into two distinct species by re versed-phase chromatography. The migration of the two mitochondrial glutamyl-tRNAs (tRNA_I^Glu and tRNA_II^Glu) differed from that of two glutamyl-tRNA species found in the cytoplasm of a mitochondrial DNA-less petite strain. Both mitochondrial tRNAs hybridized with mitochondrial DNA. Three lines of evidence demonstrate that mitochondrial tRNA_I^Glu and tRNA_II^Glu are transcribed from different mitochondrial cistrons. First the level of hybridization of a mixture of the two tRNAs to mitochondrial DNA was equal to the sum of the saturation hybridization levels of each glutamyl-tRNA alone. Second, the two mitochondrial glutamyl-tRNAs did not compete with each other in hybridization competition experiments. Finally the tRNAs showed individual hybridization patterns with different petite mitochondrial DNAs.Hybridization of the tRNAs to mitochondrial DNA of genetically defined petite strains localized each tRNA with respect to antibiotic resistance markers. The two glutamyl-tRNA cistrons were spatially separated on the genetic map.     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine,an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNA^Glu or of the non-cognate tRNA^Phe is indicated by the negative values of –TΔS_b, and by the negative value of ΔC_p. On the other hand, the large negative enthalpy is the dominant contribution to ΔG_b in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNA^Phe, but the dissociation constant K _d is decreased 50-fold in the presence of tRNA^Glu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNA^Glu in the Glu-AMS•GluRS•tRNA^Glu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNA^Glu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.     

Enzymatic Activities for the Synthesis of Chlorophyll in Pigment-Deficient Variegated Leaves of Euonymus japonicus

The enzymes involved in the biosynthesis of chlorophyll (Chl)in pigment-deficient variegated leaves of Euonymus japonicuswere investigated. Each variegated leaf was composed of clearlydelineated green and white sectors. The white sectors containedalmost no Chls. The rate of synthesis of 5-aminolevulinic acid(ALA) in the white sectors in vivo was twice that in the greensectors. The level of glutamate 1-semialdehyde aminotransferasein the white sectors was much higher than that in the greensectors. Plastidic tRNA^Glu was also present at substantial levelsin the white sectors, indicating that the system for synthesisof ALA was very active in the white sectors. The activity of porphobilinogen (PBG) synthase in the whitesectors in vitro was twice that in the green sectors. In thewhite sectors the rate of porphyrin synthesis from PBG was 4-to 6-fold higher than in the green sectors. We measured Mg-chelataseactivity indirectly in both sectors by monitoring the accumulationof Mg-protoporphyrin IX in the presence of 2,2'-dipyridyl, whichinhibits isocyclic ring formation with the resultant accumulationof Mg-protoporphyrin IX. When sectors were incubated in darknesswith 2,2'-dipyridyl, large amounts of protoporphyrin IX accumulatedin the white sectors, whereas Mg-protoporphyrin IX mainly accumulatedin the green sectors. These results suggest that the enzymesfor the synthesis of porphyrin that catalyze conversion of ALAto protoporphyrin IX were very active and that the Mg-insertionstep might be blocked in the white sectors, with the resultantfailure to synthesize Chl. The deficiency is discussed in acomparison with that in other Chl-deficient plants. (Received November 15, 1995; Accepted March 21, 1996)     

Physical and kinetic interactions between glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase of Chlamydomonas reinhardtii

In plants, algae, and most bacteria, the heme and chlorophyll precursor 5-aminolevulinic acid (ALA) is formed from glutamate in a three-step process. First, glutamate is ligated to its cognate tRNA by glutamyl-tRNA synthetase. Activated glutamate is then converted to a glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR) in an NADPH-dependent reaction. Subsequently, GSA is rearranged to ALA by glutamate-1-semialdehyde aminotransferase (GSAT). The intermediate GSA is highly unstable under physiological conditions. We have used purified recombinant GTR and GSAT from the unicellular alga Chlamydomonas reinhardtii to show that GTR and GSAT form a physical and functional complex that allows channeling of GSA between the enzymes. Co-immunoprecipitation and sucrose gradient ultracentrifugation results indicate that recombinant GTR and GSAT enzymes specifically interact. In vivo cross-linking results support the in vitro results and demonstrate that GTR and GSAT are components of a high molecular mass complex in C. reinhardtii cells. In a coupled enzyme assay containing GTR and wild-type GSAT, addition of inactive mutant GSAT inhibited ALA formation from glutamyl-tRNA. Mutant GSAT did not inhibit ALA formation from GSA by wild-type GSAT. These results suggest that there is competition between wild-type and mutant GSAT for binding to GTR and channeling GSA from GTR to GSAT. Further evidence supporting kinetic interaction of GTR and GSAT is the observation that both wild-type and mutant GSAT stimulate glutamyl-tRNA-dependent NADPH oxidation by GTR.