Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

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     

The biosynthesis of δ-aminolevulinic acid in greening maize leaves

In greening maize leaves δ-aminolevulinic acid (ALA) was not formed from succinyl-CoA and glycine as shown by the incorporation of [¹⁴C]-labeled     

Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

Generation of active oxygen species during coupled autoxidation of oxyhemoglobin and δ-aminolevulinic acid

The conversion of oxyhemoglobin to mathemoglobin has been shown via spectrophotometric, circular dichroism and polarographic studies to be accelerated by δ-aminolevulinic acid, a major heme-precursor accumulated in a number of heme-linked pathologies. Concomitantly, δ-aminolevulinic acid undergoes aerobic oxidation. The intermediacy of oxygen radicals in these processes was evidenced by the inhibitory effect of catalase, superoxide dismutase and mannitol. These results are relevant to the exacerbated production of active oxygen species in intermittent acute porphyria and saturnism carriers.     

Detection and regulation of δ-aminolevulinic acid synthetase activity in rat skeletal muscle

The presence of δ-aminolevulinic acid synthetase (ALAS) in mitochondria obtained from rat skeletal muscles has been observed. Optimal conditions for the meausurement of this activity are described. The activity of skeletal muscle ALAS was investigated under conditions known to affect the activity of this enzyme in other tissues. ALAS activity in skeletal muscle mitochondria was decreased 55% by a 48-h fast. Treatment with dexamethasone did not reverse the effect of starvation on ALAS activity and did not change the activity in the fed controls. ALAS activity was decreased 56% in skeletal muscle mitochondria obtained from rats in which diabetes mellitus had been induced by streptozotocin. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirm our previous suggestion that ALAS activity is regulated in a tissue-specific manner.     

Metabolic engineering of microorganisms for the production of multifunctional non-protein amino acids: γ-aminobutyric acid and δ-aminolevulinic acid

Gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non-protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.     

Enhanced production of δ-aminolevulinic acid, bilipigments, and antioxidants from tropical algae of India

We studied the enhanced production of high quality biomass, δ-aminolevulinic acid (δ-ALA), bilipigments, and antioxidants from five tropical blue green algae (cyanobacteria) in a full factorial design using free and immobilized cells in batch culture. Production of nutraceuticals was high in spray dried powder prepared from immobilized cell cultures. Nostochopsis lobatus showed superiority over rest of the species with respect to bilipigments, δ-ALA, nutritive value, antioxidant capacity, and ascorbate oxidase (APX) activity. Antioxidative capacity of phycobiliproteins extracted from these cyanobacteria (121.15 μM TE/g, Nostoc verrucosum to 217.62 μM TE/g, Nostochopsis lobatus) was invariably higher than those observed for higher plant sources and substantially increased under immobilized cell culture condition. Antioxidative enzyme, ascorbate oxidase remained stable in dry food preparations with considerably high activity under immobilized cell preparations (APX_max, 3.40 μmol/min/mg chlorophyll). These observations have important connotations in light of upcoming food and nutraceutical industries in the global market. Use of immobilized cells in batch culture could be an effective approach for scaling up production for commercial use.     

Cadmium induced oxidative stress in soybean plants also by the accumulation of δ-aminolevulinic acid

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 μM Cd²⁺ for 48 h. δ-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-l-methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd²⁺, it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd²⁺ and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd²⁺ is highly responsible for oxidative stress generation in these tissues.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

Evidence for histidine as another functional group of δ-aminolevulinic acid dehydratase from beef liver

δ-Aminolevulinic acid dehydratase (EC 4.2.1.24) was obtained in highly purified form from beef liver. Upon photooxidation of the enzyme in the presence of methylene blue as a sensitizer led to a loss of the enzymatic activity according to pseudo-first order kinetics. The pronounced pH dependence (pk value of 6.8) of the photooxidation rate and the results of amino acid analysis suggested that the inactivation was largely due to the modification of the histidine residue. The finding of the enzyme with little activity in the presence of diethylpyrocarbonate was consistent with such a speculation. On the basis of these results, it can be postulated that the histidine residue seems to play an important role in the enzymatic activity of δ-aminolevulinic acid dehydratase.     

Developmental and circadian control of the capacity for δ-aminolevulinic acid synthesis in green barley

The synthesis of δ-aminolevulinic acid (δ-ALA) is a key step in the regulation of tetrapyrrole synthesis. To study the developmentally and circadian-clock controlled mechanism that co-ordinates synthesis of chlorophylls and chlorophyll-binding proteins, δ-ALA-synthesising capacity was analysed in barley (Hordeum vulgare L.) primary leaves grown under dark/light or constant light conditions. The δ-ALA-forming activity oscillated within 24 h with a maximum at the transition of dark to light and a minimum 12 h later, indicating the involvement of the circadian oscillator during development. The capacity for δ-ALA synthesis increased transiently in the middle of barley primary leaves. The δ-ALA-forming-activity correlated well with the previously published steady-state level of mRNA for light-harvesting chlorophyll-binding proteins in space and time; this supports the view of a co-ordinate synthesis of chlorophyll and pigment-binding proteins. Steady-state levels of mRNAs encoding the three enzymes of the δ-ALA-synthesising pathway and of proteins for glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSA AT; EC 5.4.3.8) were analysed for their developmental and circadian expression in barley leaves. The contents of GluTR mRNA and protein cycled parallel to the changes in δ-ALA-forming activity. The levels of GSA AT mRNA oscillated in an opposite phase, but the protein content did not show substantial oscillation under diurnal and circadian growth conditions. No circadian oscillation was detected for glutamyl tRNA synthase (GluRS; EC 6.1.1.17). Maximal GluTR mRNA content and protein was observed in the middle (segments 3 and 4) of the barley primary leaves. The developmentally controlled expression of GluTR therefore differs from that of GSA AT and GluRS, but resembles the capacity for δ-ALA synthesis in a barley leaf gradient. These data indicate that the oscillating, light-dependent and spatial expression of GluTR mRNA might contribute to the regulated formation of the chlorophyll precursor δ-ALA. Received: 29 April 1996 / Accepted 11 December 1996     

Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains—Proposal of new biosynthetic pathways

Our studies of the microbial-metabolism of triterpenoid ursolic acid by various Nocardia sp. strains, have led to the proposal of two novel pathways to produce triterpenoid derivatives. Nocardia sp. NRRL 5646, Nocardia sp. 44822 and Nocardia sp. 44000 generated the following ursolic acid derivatives: ursolic acid methyl ester, ursonic acid, ursonic acid methyl ester, 3-oxoursa-1,12-dien-28-oic acid and 3-oxoursa-1,12-dien-28-oic acid methyl ester. Nocardia sp. 45077 synthesized ursonic acid and 3-oxoursa-1,12-dien-28-oic acid while Nocardia sp. 46002 produced only ursonic acid and Nocardia sp. 43069 showed no metabolism at all. The conversion of ursolic acid by Nocardia sp. NRRL 5646 was independent of the medium used for the fermentation. An increase in temperature from 28 °C to 36 °C doubled the reaction rate of the biotransformation. The analysis of ursane metabolites was done by HPLC, while their structures were established using HPLC–APCI_pos-MS/MS and HPLC–NMR spectroscopy. The pseudo molecular ion peaks were determined by HPLC–APCI_pos-MS and used to measure their molecular weight. The product ion spectra of the metabolites showed the characteristic fragments of Δ¹²-oleanes and Δ¹²-ursanes indicating that a substitution in ring A or B was responsible for the decrease in molecular weight.Based on these results, two new biosynthetic pathways are proposed. These new pathways can presumably be used as strategic routes for the biotechnological production of triterpenoid derivatives. It is assumed that a 3β-hydroxysteroid dehydrogenase and a 3-ketosteroid-Δ¹-dehydrogenase are involved in the transformation of the steroid.     

Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12

δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B₁₂, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B₁₂, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B₁₂ with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized.