Formation of delta-Aminolevulinic Acid from Glutamic Acid in Algal Extracts : Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to δ-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg²⁺, and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH₄)₂SO₄ at 70% saturation. The proteins recovered from the (NH₄)₂SO₄ precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH₄)₂SO₄ precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography.     

delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis

γ,δ-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to δ-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37° C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W₁₄ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 ± 3,000. l-Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K_m values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 ± 1 nanomoles ALA per 30 minutes per 10⁷ cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena.     

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor δ-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°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-[¹⁴C]glutamate and 3,4-[³H]glutamate but not from 1-[¹⁴C]glutamate, and thus, operation of the C₅ pathway could not be confirmed.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.     

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris

An enzyme catalyzing the formation of δ-aminolevulinic acid by transamination of γ,δ-dioxovaleric acid with l-α-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in δ-aminolevulinic acid metabolism in plants is not clearly understood.     

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.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues

δ-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. δ-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of δ-aminolevulinic acid dehydrase. The rate of δ-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of δ-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, δ-aminolevulinic acid accumulation ceased.     

Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts

Formation of pectolytic enzyme system in Erwinia aroideae was stimulated to a remarkable extent when the cells were incubated in a pectin medium containing carrot extracts. The active principle in the carrot extract preparation was resistant to acid hydrolysis, digestion by Pronase, RNase, DNase or α-amylase, and to ninhydrin and charcoal treatments. The factor lost, however, its stimulating activity upon alkaline hydrolysis or periodate oxidation. The factor was partially purified by the combination of gel filtration with Sephadex G-10 and ninhydrin and charcoal treatments. The molecular weight of the partially purified factor was presumed to be around 400 by the gel filtration.     

Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts

Formation of pectinase system in Erwinia aroideae was stimulated to a considerable extent when the cells were incubated in a pectin medium containing carrot extracts. The active factor in the extract was purified about 30 fold by ethanol precipitation, and further purification was achieved by ninhydrin treatment, charcoal adsorption, dialysis and gel filtration with Sephadex G-10. Although crude carrot extract preparation also stimulated protease formation in this organism, no stimulating activity for protease formation was found in the purified factor. Acetate and butyrate which had been shown to stimulate pectinase formation, were found to stimulated protease formation as well. Pectinase formation by this organism was also stimulated by polyamines and inorganic phosphate to a considerable extent.     

Biosynthesis of delta-Aminolevulinic Acid from Glutamate in Agmenellum quadruplicatum

δ-Aminolevulinic acid accumulated in the culture medium when Agmenellum quadruplicatum strain PR-6 was incubated in the presence of levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydratase, and specifically labeled glutamate and glycine. The δ-aminolevulinic acid was purified using Dowex 50W-X8 and cleaved by periodate to yield succinic acid and formaldehyde. The distribution of radioactivity in the two fragments suggested that in blue-green algae the carbon skeleton of δ-aminolevulinic acid is derived directly from glutamate. However the possibility of the pathway of δ-aminolevulinic acid synthesis, from glycine and succinyl-coenzyme A also functioning in blue-green algae was not eliminated as uptake of glycine was minimal.     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues

δ-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various ¹⁴C-labeled precursors and levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase. The radioactivity in the accumulated δ-aminolevulinic acid was measured.     

The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta-Aminolevulinic Acid

Etiolated cucumber cotyledons treated with δ-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the δ-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.     

Stimulation of Bacterial DNA Synthesis by Algal Exudates in Attached Algal-Bacterial Consortia

Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus [W. E. C. Moore, E. P. Cato, and L. V. H. Moore, Int. J. Syst. Bacteriol. 35:382-407, 1985]). The organisms were attached to the surfaces at cell densities of approximately 5 × 10⁴ cells cm^-2 (diatoms) and 5 × 10⁶ cells cm^-2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [³H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [³H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 × 10^-21 to 17.9 × 10^-21 mol of [³H]thymidine incorporated cell^-1 h^-1) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon.     

Studies on the Biosynthesis and Metabolism of delta-Aminolevulinic Acid in Chlorella

The regulation of chlorophyll synthesis in Chlorella was examined at the level of the formation and metabolism of δ-aminolevulinic acid. δ-Aminolevulinic acid synthetase activity could not be detected in broken cell preparations, and exogenously supplied δ-aminolevulinic acid was taken up only in the presence of dimethylsulfoxide, with a corresponding production of porphobilinogen.     

Biosynthesis of Tetrapyrrole Pigment Precursors : Formation and Utilization of Glutamyl-tRNA for delta-Aminolevulinic Acid Synthesis by Isolated Enzyme Fractions from Chlorella Vulgaris

The universal tetrapyrrole precursor δ-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 Mg²⁺. 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.     

Fatty Acid Hydroxamate Formation and Ester Hydrolysis by Cell-free Extracts of Micrococcus sp.

When Micrococcus sp. which was isolated from soil assimilated azelaic acid as a sole carbon source, cell-free extract of the organism catalyzed enzymic fatty acid hydroxamate formation. The enzyme was effective only for mono-carboxylic acid, but not for di-carboxylic acids such as azelaic acid. The activity was high with higher fatty acid such as oleic acid. Some of the properties of higher fatty acid hydroxamate formation were investigated.

Olelylhydroxamate was formed with the high concentration of hydroxylamine. The reaction was inhibited by PCMB, but recovered by the addition of SH-compounds (such as cysteine).

On the other hand, when methylacetate was used as a sole carbon source and cell-free extract of Micrococcus sp. hydrolyzed several fatty acid esters. The fatty acid hydroxamate degradation by esterolysis are also discussed.     

delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity

δ-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.

Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.

In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t_1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.

Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.

In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t_1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O₂ production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.

The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route.

     

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.