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.     

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     

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.     

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.     

Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus

Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61 kDa protein with activity of 24.3 U/mg and the OD₆₀₀ of 23 was reached after 18 h induction with 10 mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50 U/ml SSbgly and 90 mg/ml Ste at 75 °C as determined by the response surface method.     

Characterization of a β-glucosidase from Sulfolobus solfataricus for isoflavone glycosides

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K _m of 0.5 mM, and a k _cat of 2532 s⁻¹. The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h⁻¹. Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k _cat, k _cat/K _m, conversion yield, and productivity in the hydrolysis of daidzin.     

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     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.     

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.     

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.     

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.     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

An additional glucose dehydrogenase from Sulfolobus solfataricus: fine-tuning of sugar degradation?

Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner-Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61% amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency. In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways.     

Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments

Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.     

Efficient chemoselective synthesis of 3,4′-dihydroxypropiophenone 3-O-β-D-glucoside by thermophilic β-glycosidase from Sulfolobus solfataricus

Summary A comparison of different systems for the -glycosidase-atalyzed synthesis of 3,4-dihydroxypropiophenone 3-O--D-glucoside is reported including various enzymatic sources and different reaction conditions. The best yield was obtained using thermophilic -glycosidase from Sulfolobus solfataricus.     

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.     

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.