Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool

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.     

Extracellular formation of 5-aminolevulinic acid by intact cells of the marine photosynthetic bacterium Rhodovulum sp. under various pH conditions

Extracellular formation of 5-aminolevulinic acid (ALA) by Rhodovulum sp. PS88 correlated with the consumption of the undissociated form of levulinic acid (LA) in an intact cell system. The concentration of the undissociated form of LA governed the extracellular formation of ALA at various culture pH values. This phenomenon might be caused by inhibition of ALA dehydratase by the undissociated form of LA after uptake into the cells as observed in Rhodobacter sphaeroides.     

5-Aminolevulinic acid production by Chlorella sp. during heterotrophic cultivation in the dark

5-Aminolevulinic acid (ALA) was produced aerobically in the dark during growth on glucose by a newly isolated Chlorella sp. When levulinic acid (20 mM), a competitive inhibitor of ALA dehydratase, was added repeatedly to the medium, about 1.5 mM of ALA was produced extracellularly. Glutamate (30 mM) added with levulinic acid (20 mM, given repeatedly) enhanced ALA production up to 1.9 mM, indicating that ALA might be synthesized via the C-5 pathway.K. Sasaki was with the Hiroshima-Denki Institute of Technology, 6-20-1, Nakano, Akiku, Hiroshima, 739-03, Japan; and is now with the Department of Biotechnology. The University of New South Wales, Sydney, NSW, 2052, Australia; K. Watanabe, T. Tanaka and Y. Hotta are with the Cosmo Research Institute, 1134-2, Gongendo, Satte, Saitama, 340-01, Japan. S. Nagai is with the Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 4-1, Kagamiyama 1 chome, Higashi-Hiroshima, 724. Japan.     

Enhanced production of 5-aminolevulinic acid by repeated addition of levulinic acid and supplement of precursors in photoheterotrophic culture of Rhodobacter sphaeroides

When levulinic acid (LA) was added repeatedly in the photosynthetic culture of Rhodobacter sphaeroides to maintain its concentration at a constant (low) level, the activity of 5-aminolevulinic acid (ALA) dehydratase could be kept low enough to reduce the synthesis of porphobilinogen from ALA formed. This brought about the extracellular accumulation of ALA, while cellular growth was partially retarded. In this condition, the intermittent or continuous supplement of a fixed amount of the pre-cursors (glycine and succinate) to reduce the inhibitory effects of glycine on growth could enhance the ALA accumulation twice as much as when the same amount of the precursors was supplied all at once.     

Production of 5-aminolevulinic acid by photosynthetic bacteria

Extracellular formation of 5-aminolevulinic acid (ALA) by adding levulinic acid (LA), an inhibitor of ALA dehydratase, was examined in the anaerobic-light culture of Rhodobacter sphaeroides. The addition of LA (10–25 mmol/l) during the middle log phase retarded the growth and accelerated the extracellular formation of ALA, while over 50 mmol/l completely suppressed both growth and formation.The formation of ALA was closely related to intracellular ALA synthetase activity. Light intensity was also an important factor for enhancing ALA formation. The optimal condition, addition of 15 mmol/l of LA during the middle log phase with 3 klx illumination, resulted in ALA formation of 0.26 mol/l. In addition, supplementation with glycine (30 mmol/l) and succinate (30 mmol/l), precursors of ALA biosynthesis, enhanced ALA formation up to ca. 2 mmol/l.     

Genetic control of chlorophyll biosynthesis by the plastome in some Oenothera species (subgenus Munzia)

Reciprocal differences in the rates of chlorophyll (Chl) formation during early stages of greening are observed in hybrid seedlings with identical genomes derived from reciprocal crosses between Oenothera berteriana (=villaricae) and Oe. odorata (=picensis), subgenus Munzia. In the presence of levulinic acid (LA), a competitive inhibitor of 5-aminolevulinic acid (ALA) dehydratase, ALA accumulated in the cotyledons and chlorophyll production was reduced in a stoichometric ratio. Accumulation of both Chl in untreated tissue and of ALA in seedlings incubated with LA is much more rapid in cotyledons with berteriana plastids than in those with odorata plastids. No difference was found between the inhibitor constants for LA of ALA dehydratase extracted from seedlings with either berteriana or odorata plastids. ALA formation is not limited by the availability of possible precursors. ALA dehydratase and the porphobilinogenase complex (PBGase) are present in abundance and in equal amounts in cotyledons with either berteriana or odorata plastids. It is concluded that the different capacities of the ALA synthesizing system fully account for the different rates of Chl formation in the seedlings with identical genomes and different plastid types.Abbreviations Chl chlorophyll - ALA 5-aminolevnlinic acid - ALAD 5-aminolevulinic acid dehydratase - LA levulinic acid - PBG porphobilinogen - PBGase porphobilinogenase - Oe Oenothera - bert berteriana - od odorata - Pl plastids     

Optimization of extracellular 5-aminolevulinic acid production from Escherichia coli transformed with ALA synthase gene of Bradyrhizobium japonicum

Extracellular accumulation of 5-aminolevulinic acid (ALA) by an E. coli overexpressing ALA synthase (ALAS) was achieved by inserting a hemA gene from Bradyrhizobium japonicum and expressed under the control of T7 promoter. At pH 7.0 extracellular ALA reached up to 15 mM in a jar fermenter with an addition of glycine (30 mM) and succinate (90 mM) in the medium. ALA accumulation was increased to 20 mM by adding levulinic acid (30 mM) to the cultures.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.     

Formation of 5-aminolevulinic acid under aerobic/dark condition by a mutant ofRhodobacter sphaeroides

Summary A mutant strain CR-17 ofRhodobacter sphaeroides was characterized by the low activity of 5-aminolevulinic acid dehydratase (ALAD) compared to the wild-type strain. Without addition of levulinic acid (inhibitor of ALAD), the mutant secreted 5-aminolevulinic acid under anaerobic/light (64.5 M) or under microaerobic/dark (32 M) conditions; 82.5 M of ALA was secreted under aerobic/dark condition with addition of 15 mM levulinic acid.     

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.     

Modulation of cytochrome biosynthesis in yeast by antimetabolite action of levulinic acid.

Levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was used to inhibit cytochrome biosynthesis in growing yeast cells. In Saccharomyces cerevisiae the antimetabolite acts by inhibiting delta-aminolevulinic acid dehydratase in vivo, causing an accumulation of intracellular delta-aminolevulinic acid and simultaneous decreases in all classes of mitochondrial cytochromes. Changes in cellular cytochrome content with increasing levulinic acid concentration suggested the existence of different regulatory patterns in S. cerevisiae and Candida utilis. In C. utilis, cytochrome a.a3 formation is very resistant to the antimetabolite action of levulinic acid. In this aerobic yeast, cytochrome c+c1 is the most sensitive to levulinic acid, and cytochrome b exhibits intermediate sensitivity.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.     

Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme content and delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10^?7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.     

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.     

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。     

Effect of culture pH on the extracellular production of 5-aminolevulinic acid by Rhodobacter sphaeroides from volatile fatty acids

Summary 5-Aminolevulinic acid(ALA) production by Rhodobacter sphaeroides was investigated at various pH with levulinic acid addition using a volatile fatty acids medium prepared from the mandarin orange peel supplemented with glycine. At neutral pH (6.8 and 7.0), extracellular ALA production was up to 16 mM, while low production of ALA(less than 3.5 mM) was observed at acidic pH (lower than 6.5) and less than 3.9 mM of ALA produced at alkaline pH (higher than 7.5). The higher ALA synthase activity observed at neutral pH might enhance the ALA production compared with that observed in acidic and alkaliphilic cultures.     

Biosynthesis of delta-aminolevulinic acid by blue-green algae (cyanobacteria).

When levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was added to growing cultures of blue-green algae (cyanobacteria), delta-aminolevulinic acid was excreted into the medium and cell growth was inhibited.     

Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme

Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells. Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs. A set of overlapping cDNAs was characterized that encode the pea enzyme. The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases. The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases. Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate. Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots. Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.