ALA Formation Research Articles

Biosynthesis of ?-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the α-group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the α-proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-14C]glutamate, but not from [2-14C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.

Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis.

5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles, such as chlorophyll and heme. The major control of chlorophyll biosynthesis is at the step of ALA formation. In the chloroplasts of plants, as in Escherichia coli, ALA is derived from the glutamate of Glu-tRNA via the two-step C5 pathway. The first enzyme, Glu-tRNA reductase, catalyzes the reduction of Glu-tRNA to glutamate 1-semialdehyde with the release of intact tRNA. The second enzyme, glutamate 1-semialdehyde 2,1-aminomutase, converts glutamate 1-semialdehyde to ALA. To further examine ALA formation in plants, we isolated Arabidopsis genes that encode the enzymes of the C5 pathway via functional complementation of mutations in the corresponding genes of E. coli. The Glu-tRNA reductase gene was designated HEMA and the glutamate 1-semialdehyde 2,1-aminomutase gene, GSA1. Each gene contains two short introns (149 and 241 nucleotides for HEMA, 153 and 86 nucleotides for GSA1). The deduced amino acid sequence of the HEMA protein predicts a protein of 60 kD with substantial similarity (30 to 47% identity) to sequences derived from the known hemA genes from microorganisms that make ALA by the C5 pathway. Purified Arabidopsis HEMA protein has Glu-tRNA reductase activity. The GSA1 gene encodes a 50-kD protein whose deduced amino acid sequence shows extensive homology (55 to 78% identity) with glutamate 1-semialdehyde 2,1-aminomutase proteins from other species. RNA gel blot analyses indicated that transcripts for both genes are found in root, leaf, stem, and flower tissues and that their levels are dramatically elevated by light. Thus, light may regulate ALA, and hence chlorophyll formation, by exerting coordinated transcriptional control over both enzymes of the C5 pathway.

Incomplete citric acid cycle obliges aminolevulinic acid synthesis via the C5 pathway in a methylotroph

The enzymic activities of the citric acid cycle and the connected pathway of 5-aminolevulinic acid (ALA) formation in the methylotroph Methylophilus methylotrophus (strain AS1) have been studied. The organism has the enzymes required for conversion of pyruvate to 2-oxoglutarate. Of these, isocitrate dehydrogenase is unusual because of its preference of NAD as coenzyme over NADP. In addition, the segment of the cycle that oxidizes 2-oxoglutarate to oxaloacetate is incomplete, lacking 2-oxoglutarate and succinate and malate dehydrogenase activities. Furthermore, alternative routes of 2-oxoglutarate oxidation to succinate are undetectable. The enzymes of the glyoxylate cycle are also absent. This suggests that the cycle in M. methylotrophus has no catabolic role and is purely biosynthetic. We also show that M. methylotrophus uses the C5 pathway of ALA formation. Cell-free extracts can convert glutamate to ALA in an ATP-, NADPH- and tRNA-dependent manner via the intermediate formation of Glu-tRNAGlu and glutamate 1-semialdehyde. Consistent with the absence of a detectable route by which it could synthesize succinate, M. methylotrophus cannot generate ALA from succinyl-CoA and glycine, the pathway found in mammalian cells and yeast.

The role of ALA‐S and ALA‐D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae

Catabolite repression and derepression on delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated. ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.

Succinyl-Coenzyme A Synthetase and its Role in δ-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, delta-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain alpha-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, alpha-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and alpha-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP(+), and was inhibited by NAD(+). Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of alpha-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and Söll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.

Characterization of δ-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor delta-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1-[(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed.

δ-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, delta-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 W(14)ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA(Glu). Other cellular tRNA(Glu)s 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.

Separation and partial characterization of enzymes catalyzing δ-aminolevulinic acid formation in Synechocystis sp. PCC 6803

Formation of the universal tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differential ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 μ m, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 μ m. GSA aminotransferase was inhibited 50% by 0.5 μ m gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 °C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.

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.

Characterization of the process of 5-aminolevulinic acid formation from glutamate via the C 5 pathway in Clostridium thermoaceticum

1. 1. In vitro formation of 5-aminolevulinic acid (ALA) from glutamate required two enzyme fractions, separable on Blue Sepharose affinity chromatography, and a tRNA fraction, which can be replaced by Escherichia coli tRNA Glu in the reconstituted assay. 2. 2. Gabaculine was shown to inhibit ALA formation in the complete assay as well as in a defined system consisting of only glutamate-1-semialdehyde and the enzyme fraction not retained on Blue Sepharose. 3. 3. The results indicate that the enzyme system supporting ALA formation in Clostridium thermoaceticum is very similar to the tRNA Glu-dependent C 5 pathway in plant plastids.

Delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Light Regulation of δ-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, delta-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.

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

delta-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 alpha-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.

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts

Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described.

Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Chlorobium vibrioforme

Formation of the tetrapyrrole pigment precursor delta-aminolevulinic acid (ALA) from glutamate was detected and partially characterized in extracts of the strictly anaerobic green photosynthetic bacterial species Chlorobium vibrioforme by using assay methods derived from those developed for algae and cyanobacteria. ALA formation in Chlorobium extracts was saturated at 10 mM glutamate and required NADPH and ATP at optimal concentrations of 0.3 and 3 mM, respectively. Preincubation of the enzyme extract with RNase A destroyed the ALA-forming activity completely. Activity in the RNase-treated extract was restored by supplementation with Chlorobium RNA after addition of RNasin to block further RNase action. RNA from the cyanobacterium Synechocystis sp. strain PCC 6803 and Escherichia coli tRNAGlu also restored activity. Activity was inhibited 50% by 0.2 microM hemin. ALA formation was completely abolished by the addition of 5 microM 3-amino-2,3-dihydrobenzoic acid (gabaculine). These results indicate that Chlorobium extracts share with those of plants, eucaryotic algae, cyanobacteria, prochlorophytes, and methanogens the capacity for RNA-dependent ALA formation from glutamate.

Inhibition of Bacteriochlorophyll Biosynthesis by Gabaculin (3-Amino, 2,3-dihydrobenzoic Acid) and Presence of an Enzyme of the C5-Pathway of δ-Aminolevulinate Synthesis in Chloroflexus aurantiacus

Abstract Biosynthesis of bacteriochlorophyll c and a in the thermophilic phototrophic prokaryote. Chloroflexus aurantiacus Ok-70-fl, was strongly inhibited by the antibiotic gabaculin (3-amino 2,3- dihydrobenzoic acid), an inhibitor of the glutamate-C5-pathway of ö-aminolevulinate (ALA) synthesis. The key enzyme of the Shemin-pathway of ALA formation, ALA synthase (EC 2.3.1.37), was not detected in cell extracts of Chi. aurantiacus. However, the extracts catalyzed ALA formation from glutamate 1-semialdehyde, a reaction being highly sensitive to gabaculin.

The tRNA Required for in Vitro δ-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts

RNA is an essential component for the enzymic conversion of glutamate to delta-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 [(3)H]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 [(3)H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.

Biosynthesis of Tetrapyrrole Pigment Precursors

The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg(2+). The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 +/- 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process.

Characterization of the RNA Required for Biosynthesis of δ-Aminolevulinic Acid from Glutamate

The heme and chlorophyll precursor delta-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3' terminus of yeast tRNA(Phe(GAA)) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA(Glu(UUC)) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A(260) unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae.