5-氨基乙酰丙酸的光动力应用研究进展

对光合细菌、藻类及其它细菌的5-氨基乙酰丙酸(5-ALA)产量进行了对比,类球红细菌(Rhodobacter sphaeoides)在黑暗、厌氧条件下培养,可产生大量的胞外5-ALA。5-ALA在农业生产中作为光动力除草剂、杀虫剂取得了很好效果。并且,ALA对提高植物的抗盐、抗冷冻能力也有一定作用。近年来,ALA在癌症治疗、肿瘤诊断方面也得到了广泛应用。     

光合细菌嗜酸柏拉红菌5-氨基乙酰丙酸合成酶基因的克隆与原核表达

5-氨基乙酰丙酸(ALA)可作为除草剂、杀虫剂和植物生长调节剂在农业上应用,但由于其成本较高而限制了它的大面积使用。利用常规基因工程操作方法结合载体介导PCR法(Vecterette PCR)克隆了嗜酸柏拉红菌(Rhodoblastus acidophilus)的5-氨基乙酰丙酸合成酶(ALAS)基因。并将编码ALAS的基因插入到原核表达载体pQE30中,在大肠杆菌不同菌株(E.coli JM109、M15及BL21(DE3))中进行诱导表达。对产物进行SDS-PAGE分析表明,ALAS基因已在细菌中成功表达。使用Ni-NTA亲和层析法对表达的ALAS进行分离、纯化,得到大小约为44kD的ALAS蛋白。通过优化工程菌株的培养条件,建立了发酵生产ALA的方法,其胞外分泌ALA产量达5.379g/L,ALAS酶活力高达333U/min.mg。这是目前国内外利用生物法生产ALA产量最高的报道,为ALA的产业化应用打下了良好的基础。     

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.     

Omega-3 fatty acids and antioxidants in edible wild plants

Human beings evolved on a diet that was balanced in the omega-6 and omega-3 polyunsaturated fatty acids (PUFA), and was high in antioxidants. Edible wild plants provide alpha-linolenic acid (ALA) and higher amounts of vitamin E and vitamin C than cultivated plants. In addition to the antioxidant vitamins, edible wild plants are rich in phenols and other compounds that increase their antioxidant capacity. It is therefore important to systematically analyze the total antioxidant capacity of wild plants and promote their commercialization in both developed and developing countries. The diets of Western countries have contained increasingly larger amounts of linoleic acid (LA), which has been promoted for its cholesterol-lowering effect. It is now recognized that dietary LA favors oxidative modification of low density lipoprotein (LDL) cholesterol and increases platelet response to aggregation. In contrast, ALA intake is associated with inhibitory effects on the clotting activity of platelets, on their response to thrombin, and on the regulation of arachidonic acid (AA) metabolism. In clinical studies, ALA contributed to lowering of blood pressure, and a prospective epidemiological study showed that ALA is inversely related to the risk of coronary heart disease in men. Dietary amounts of LA as well as the ratio of LA to ALA appear to be important for the metabolism of ALA to longer-chain omega-3 PUFAs. Relatively large reserves of LA in body fat. as are found in vegans or in the diet of omnivores in Western societies, would tend to slow down the formation of long-chain omega-3 fatty acids from ALA. Therefore, the role of ALA in human nutrition becomes important in terms of long-term dietary intake. One advantage of the consumption of ALA over omega-3 fatty acids from fish is that the problem of insufficient vitamin E intake does not exist with high intake of ALA from plant sources.     

Selective inhibition of HEMA gene expression by photooxidation in Arabidopsis thaliana.

Norflurazon (NF), a photobleaching herbicide, inhibits carotenoid biosynthesis. Lack of carotenoid pigments leads to photooxidative damage of chloroplasts. In this study of Arabidopsis thaliana we demonstrate that NF-treated photobleached plants are still able to make 5-aminolevulinic acid (ALA) the first precursor of porphyrins and tetrapyrroles. ALA is formed in the tRNA-dependent two-step C5-pathway in the chloroplast of plants. The expression of glutamyl-tRNA reductase (GluTR), the first enzyme in the pathway, was severely inhibited by NF, while treatment with this compound did not significantly reduce the levels of the other enzyme, glutamate-l-semialdehyde aminomutase, or of tRNA(Glu), the initial metabolite of the pathway. Extracts of these plants retained the capacity, albeit reduced, to convert exogenously added glutamate to ALA. Thus, the much-reduced level of ALA formation in photobleached plants is due to selective inhibition of GluTR expression.     

Chilling tolerance in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases

The lipid composition of membranes is a key determinant for cold tolerance, and enzymes that modify membrane structure seem to be important for low-temperature acclimation. We have characterized ALA1 (for aminophospholipid ATPase1), a novel P-type ATPase in Arabidopsis that belongs to the gene family ALA1 to ALA11. The deduced amino acid sequence of ALA1 is homologous with those of yeast DRS2 and bovine ATPase II, both of which are putative aminophospholipid translocases. ALA1 complements the deficiency in phosphatidylserine internalization into intact cells that is exhibited by the drs2 yeast mutant, and expression of ALA1 results in increased translocation of aminophospholipids in reconstituted yeast membrane vesicles. These lines of evidence suggest that ALA1 is involved in generating membrane lipid asymmetry and probably encodes an aminophospholipid translocase. ALA1 complements the cold sensitivity of the drs2 yeast mutant. Downregulation of ALA1 in Arabidopsis results in cold-affected plants that are much smaller than those of the wild type. These data suggest a link between regulation of transmembrane bilayer lipid asymmetry and the adaptation of plants to cold.     

Intermolecular nitrogen transfer in the enzymic conversion of glutamate to delta-aminolevulinic acid by extracts of Chlorella vulgaris.

delta-Aminolevulinic acid (ALA), the universal biosynthetic precursor of tetrapyrrole pigments, is synthesized from glutamate in plants, algae, and many bacteria via a three-step process that begins with activation by ligation of glutamate to tRNA(Glu), followed by reduction to glutamate-1-semialdehyde (GSA) and conversion of GSA to ALA. The GSA aminotransferase step requires no substrate other than GSA. A previous study examined whether the aminotransferase reaction proceeds via intramolecular or intermolecular N transfer and concluded that the reaction catalyzed by Chlamydomonas extracts occurs via intermolecular N transfer (Y.-H.L. Mau and W.-Y. Wang [1988] Plant Physiol 86: 793-797). However, in that study the possibility was not excluded that the result was a consequence of N exchange among product ALA molecules during the incubation, rather than intermolecular N transfer during the conversion of GSA to ALA. Therefore, this question was reexamined in another species and with additional controls. A gel-filtered extract of Chlorella vulgaris cells was incubated with ATP, Mg2+, NADPH, tRNA, and a mixture of L-glutamate molecules, one-half of which were labeled with 15N and the other half with 13C at C-1. The ALA product was purified, derivatized, and analyzed by gas chromatography-mass spectrometry. A significant fraction of the ALA molecules was heavy by two mass units, indicating incorporation of both 15N and 13C. These results show that the N and C atoms of each ALA molecule were derived from different glutamate molecules. Control experiments indicated that the results could not be attributed to exchange of N atoms between glutamate or ALA molecules during the incubation. These results confirm the earlier conclusion that GSA is converted to ALA via intermolecular N transfer and extend the results to another species. The labeling results, combined with the results of kinetic and inhibitor studies, support a model for the GSA aminotransferase reaction in which a single molecule of GSA is converted to ALA via an enzyme-bound 4,5-diaminovaleric acid intermediate.     

Molecular diversity and hydrolytic enzymes production abilities of soil bacteria

Soil is an integral part of ecosystem which is niche for varieties of microflora. The present study was investigated to isolate varied strains of bacteria from soil samples of three different geographical regions of Tamil Nadu (India) and evaluate their hydrolytic enzymes (amylase, cellulase, and inulinase) producing potentialities. Among 72 bacterial cultures isolated from Ambattur Industrial Estate, Neyveli Lignite Corporation, and Arignar Anna Zoological Park regions, 41.66, 38.88, and 36.11% of isolates were observed amylase, cellulase, and inulinase producers, respectively. On the other hand, 20.83% of total bacteria isolated from all three regions exhibited concurrent production of amylase, cellulase, and inulinase. Potent isolates depicting maximum enzyme activities were identified as Bacillus anthracis strain ALA1, Bacillus cereus strain ALA3, Glutamicibacter arilaitensis strain ALA4, and Bacillus thuringiensis strain ALA5 based on molecular characterization tools. Further, the thermodynamics parameters, open reading frames (ORFs) regions, and guanine-cytosine (GC) content were determined by distinct bioinformatics tools using 16S rRNA sequences of strains. Minimum free energy values for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 were calculated as −480.73, −478.76, −496.63, and −479.03 kcal/mol, respectively. Mountain plot and entropy predicted the hierarchical representation of RNA secondary structure. The GC content of sequence for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 was calculated as 53.06, 52.94, 56.78, and 53.06%, respectively. Nine ORFs were obtained for strain ALA1, strain ALA3, and strain ALA5 while 10 ORFs were observed for strain ALA4. Additionally, bootstrap tree demonstrated close resemblance of strains with existing bacteria of similar genus. Findings showed higher variability of bacterial diversity as hydrolytic enzymes producers in the investigated geographical regions.     

The role of 5-aminolevulinic acid in the response to cold stress in soybean plants

In this study, the possibility of enhancing cold stress tolerance of soybean plants (Glycine max L.) by exogenous application of 5-aminolevulinic acid (ALA) was investigated. ALA was added to the Hoagland solution at various concentrations ranging from 0 to 40 μM for 12 h. After ALA treatment, the plants were subjected to cold stress at 4°C for 48 h. ALA at low concentrations (5-10 μM) provided significant protection against cold stress compared to non-ALA-treated plants, enhancing chlorophyll content (Chl) as well as relative water content (RWC). Increase of thiobarbituric acid reactive species (TBARS) levels was also prevented, whereas exposure to higher ALA concentrations (15-40 μM) brought about a dose dependent increase of these species, reaching a maximum of 117% in plants pre-treated with 40 μM ALA compared to controls. ALA pre-treatment also enhanced catalase (CAT) and heme oxygenase-1 (HO-1) activities. These findings indicate that HO-1 acts not only as the rate limiting enzyme in heme catabolism, but also as an antioxidant enzyme. The highest cold tolerance was obtained with 5 μM ALA pre-treatment. Results show that ALA, which is considered as an endogenous plant growth regulator, could be used effectively to protect soybean plants from the damaging effects of cold stress by enhancing the activity of heme proteins, e.g., catalase (CAT) and by promoting heme catabolism leading to the production of the highly antioxidant biliverdin and carbon monoxide, without any adverse effect on the plant growth.     

5-Aminolevulinic acid promotes anthocyanin accumulation in Fuji apples

5-Aminolevulinic acid (ALA) is an essential precursor of all tetrapyrrole compounds such as chlorophylls and heme in plants. It has also been suggested widely for applications to crops to enhance growth and production as a plant growth regulator. However, how successful ALA can be used in fruit production was rarely reported. We conducted a field experiment at eight locations in four provinces across eastern China; and the results showed that application of ALA solutions to ‘Fuji’ apple (Malus × domestica Borkh.) fruits 20 days prior to harvest significantly increased the anthocyanin content in the fruit skin. Also, ALA treatment increased the anthocyanin content of the detached apple skin in a growth chamber. Results from the semi-quantitive RT-PCR analysis showed that ALA induced gene expressions related to anthocyanin biosynthesis, including the structural genes Pal, Chs and Ufgt, and regulatory genes Myb, bHLH and Wd40. When levulinic acid (LA), an inhibitor of ALA dehydrase, was added, ALA promotion of anthocyanin accumulation and up-regulation of gene expressions were inhibited. Taken together, these results suggest that ALA promotion of anthocyanin accumulation in apples was facilitated by the up-regulation of gene expression, which might be related to the conversion of ALA to porphyrins.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Regulation in Plant Stress Tolerance by a Potential Plant Growth Regulator, 5-Aminolevulinic Acid

Exogenous application of different plant growth regulators is a well-recognized strategy to alleviate stress-induced adverse effects on different crop plants by regulating a variety of physiobiochemical processes such as photosynthesis, chlorophyll biosynthesis, nutrient uptake, antioxidant metabolism, and protein synthesis, which are directly or indirectly involved in the mechanism of stress tolerance. Of various environmental factors, salinity, drought, and extreme temperature (low or high) considerably diminish plant growth and yield by modulating endogenous levels as well as signaling pathways of plant hormones. Of various plant hormones/regulators, a potential plant growth regulator, 5-aminolevulinic acid (ALA), is known to be effective in counteracting the injurious effects of various abiotic stresses in plants. Until now the mechanisms behind ALA regulation of growth under stress have not been fully elucidated. It is also not yet clear how far growth and yield in different crops can be promoted by exogenous application of ALA and whether this ALA-induced growth and yield promotion is cost-effective. Thus, in this review we discuss at length the effects of ALA in regulating growth and development in plants under a variety of abiotic stress conditions, including salinity, drought, and temperature stress. Furthermore, advances in the functional and regulatory interactions of this plant growth regulator with plant stress tolerance, as well as the effective mode of exogenous application of ALA in inducing stress tolerance in plants are also comprehensively discussed in this review. In the future, overaccumulation of ALA in plants through manipulation of gene(s) could enhance plant stress tolerance. Thus, genetic manipulation of plants with the goal of attaining increased synthesis/accumulation of ALA and hence improved stress tolerance under stress conditions is an important area for research.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -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 -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 -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.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

Microbial production and applications of 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), an important intermediate in tetrapyrrole biosynthesis in organisms, has been widely applied in many fields, such as medicine, agriculture, and the food industry, due to its biochemical characteristics. Research efforts supporting the microbial production of ALA have received increasing interest due to its dominant advantages over chemical synthesis, including higher yields, lesser pollutant emissions, and a lesser monetary cost. ALA synthesis using photosynthetic bacteria (PSB) is a promising approach in various microbial synthesis methods. In this review, recent advances on the microbial production of ALA with an emphasis on PSB are summarized, the key enzymes in the biosynthesis pathway (especially the relationship between key enzymes and key genes) are detailed, regulation strategies are described, and the significant influencing factors on the ALA biosynthesis and application of ALA are outlined. Furthermore, the eco-friendly perspective involving the combination of wastewater treatment and microbial production of ALA is conceived.     

5-Aminolevulinic acid improves photosynthetic gas exchange capacity and ion uptake under salinity stress in oilseed rape (Brassica napus L.)

Salinity is one of the major constraints in oilseed rape (Brassica napus L.) production. One of the means to overcome this constraint is the use of plant growth regulators to induce plant tolerance. To study the plant response to salinity in combination with a growth regulator, 5-aminolevulinic acid (ALA), oilseed rape plants were grown hydroponically in greenhouse conditions under three levels of salinity (0, 100, and 200 mM NaCl) and foliar application of ALA (30 mg/l). Salinity depressed the growth of shoots and roots, and decreased leaf water potential and chlorophyll concentration. Addition of ALA partially improved the growth of shoots and roots, and increased the leaf chlorophyll concentrations of stressed plants. Foliar application of ALA also maintained leaf water potential of plants growing in 100 mM salinity at the same level as that of the control plants, and there was also an improvement in the water relations of ALA-treated plants growing in 200 mM. Net photosynthetic rate and gas exchange parameters were also reduced significantly with increasing salinity; these effects were partially reversed upon foliar application with ALA. Sodium accumulation increased with increasing NaCl concentration which induced a complex response in the macro-and micronutrients uptake and accumulation in both roots and leaves. Generally, analyses of macro- (N, P, K, S, Ca, and Mg) and micronutrients (Mn, Zn, Fe, and Cu) showed no increased accumulation of these ions in the leaves and roots (on dry weight basis) under increasing salinity except for zinc (Zn). Foliar application of ALA enhanced the concentrations of all nutrients other than Mn and Cu. These results suggest that under short-term salinity-induced stress (10 days), exogenous application of ALA helped the plants improve growth, photosynthetic gas exchange capacity, water potential, chlorophyll content, and mineral nutrition by manipulating the uptake of Na⁺.     

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.     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.