Cloning and Sequence Analysis of the hemB Gene of Rhodothermus marinus

A Rhodothermus marinus gene, hemB, coding for 5-aminolevulinic acid (ALA) dehydratase (ALAD) has been cloned and sequenced. The reading frame of the hemB gene is 1020 base pairs encoding a protein of 340 amino acids with a calculated molecular mass of 37.4 kDa. The amino acid sequence shows homology with eubacterial and eukaryotic ALA dehydratases. A putative metal-binding site of the protein shows strongest homology with corresponding sites from plant ALA dehydratases that require Mg²⁺ for activity. It differs with respect to only one amino acid out of 20 from a corresponding site in pea ALAD. Received: 1 March 1999 / Accepted: 7 April 1999     

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.     

Characterization of three homoeologous cDNAs encoding chloroplast-targeted aminolevulinic acid dehydratase in common wheat

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat (Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 (TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.     

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.     

Influence of zinc-saccharide complexes on some haematological parameters in rats

The effects of three recently synthesized zinc-saccharide complexes (zinc-fructose, zinc-galactose and zinc-glucose) on blood -aminolevulinic acid dehydratase (ALAD) activity, glutathione (GSH), zinc-protoporphyrin (ZPP) and urinary -aminolevulinic acid (ALA) levels have been investigated to ascertain the utility of these complexes as zinc supplements and as preventive agents against lead intoxication in rats.     

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     

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus

The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.     

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.     

Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves

Cesium chloride (CsCl) treatment of greening primary leaves of barley for 8 h inhibited chlorophyl] accumulation in a concentration-dependent manner and led to the accumulation of excessive amounts of uroporphyrin(ogen) III (URO[gen]) and to a minor extent of heptacarboxylporphyrin(ogen). When dark-grown leaves were incubated with CsCl, accumulation of URO(gen) was observed only after feeding of the tetrapyrrole precursor 5-aminolevulinic acid. Western blot analysis showed no apparent difference in content of uroporphyrinogen decarboxylase (EC 4.1.1.37, UROD) or selected proteins involved in tetrapyrrole biosynthesis in extracts of CsCl-incubated (15 m M ) versus control leaves. UROD activity was drastically decreased upon CsCl treatment in leaves incubated in the dark or in the light (44 and 86%, respectively). Selected preceding enzymes of the tetrapyrrole biosynthetic pathway, 5-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) and porphobilinogen deaminase (EC 4.3.1.8, PBGD), were influenced only to a minor extent under standard incubation conditions (15 m M CsCl). Furthermore, the ALA synthesizing capacity did not differ in leaves incubated with and without Cs⁻ cations. UROD activity of crude homogenates from control plants and after partial purification was reduced to 56 and 80%, respectively, upon addition of 10 m M CsCl. Equal concentrations of KCl were not inhibitory. Enzyme assays of the same barley extract in the presence of CsCl yielded no effect on ALAD and a minor loss of PBGD activity. The initial visible cytotoxic effect of CsCl appeared to be a selective inhibition of UROD resulting in accumulation of photosensitizing URO (gen). Consequences of the diminished UROD activity on early steps of the tetrapyrrole biosynthesis and its functional and regulatory significance for the porphyrin synthesis are discussed.     

Influence of L-lysine and zinc administration during exposure to lead or lead and ethanol in rats

Influence of lysine and zinc administration on the lead-sensitive biochemical parameters and the accumulation of lead during exposure to lead or lead and ethanol was investigated in rats. The lead exposure inhibited blood δ-aminolevulinic acid dehydratase (ALAD) activity, increased blood zinc protoporphyrin (ZPP), urinary δ-aminolevulinic acid (ALA), serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), blood and tissue lead levels, and decreased blood and hepatic glutahione (GSH) contents. Some of these effects were enhanced on coexposure to ethanol. The simultaneous administration of lysine and zinc reduced tissue accumulation of lead and most of the lead-induced biochemical alterations irrespective of exposure to lead alone or lead and ethanol. The depletion of endogenous calcium and magnesium owing to lead or ethanol exposure was also prevented by co-administration of lysine and zinc.     

Effect of Ni2+ on Early Stages of Chlorophyll Biosynthesis and Pheophytinization in Euglena gracilis

The effect of Ni²⁺ on the early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis cells was studied. Incubation of the cells with 10^–4 M Ni²⁺ for 7 days resulted in a higher chlorophyll content, enhanced production of 5-aminolevulinic acid (ALA), and in increased activity of 5-aminolevuluinic acid dehydratase (EC 4.2.1.24, ALAD), as compared to the control cells incubated without Ni²⁺. At a higher concentration (10^–3 M), Ni²⁺ markedly inhibited chlorophyll accumulation and ALAD activity, as compared to the control cells. At this concentration, Ni²⁺ also inhibited heme biosynthesis and strongly stimulated ALA production. It seems likely that, by affecting heme synthesis, Ni²⁺ increases the activity of the ALA production system. However, the suppression of subsequent stages of ALA conversion to chlorophyll, in particular ALAD inhibition, ultimately resulted in almost complete inhibition of chlorophyll biosynthesis. In addition to cessation of de novo chlorophyll synthesis in the presence of Ni²⁺ (10^–3 M) in Euglena cells, the existing chlorophyll was converted into pheophytin and almost completely degraded. We suppose that the Ni²⁺-induced pheophytinization is caused by an acidic shift of intracellular pH related to an impairment of cell membrane permeability by Ni²⁺ cations.     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

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.     

Porphyrin metabolism in lead and mercury treated bajra (Pennisetum typhoideum) seedlings

Lead and mercury inhibited porphyrin biosynthesis significantly in the germinating seeds of bajra (Pennisetum typhoideum). Both 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were inhibited by these metals. A comparative study of the inhibition of these two enzymes under invivo andin vitro conditions showed that 5-aminolevulinic acid dehydratase is the major site of action of heavy metals in porphyrin biosynthesis. Further, over-all production of porphyrinsviz., protoporphyrin-IX, Mg-protoporphyrin (ester) and protochlorophyllide was repressed by lead and mercury in both light and dark grown seedlings. Similarly, chlorophylla and chlorophyllb and total chlorophyll contents in dark-grown seedlings were also significantly decreased, suggesting the impairment of chlorophyll biosynthesis by lead and mercury in germinating seedlings.     

Molecular Analysis of δ-Aminolevulinic Acid Dehydratase (ALAD) Gene Polymorphism in a Turkish Population

delta-Aminolevulinic acid dehydratase (ALAD) is a cytosolic enzyme in the heme biosynthetic pathway. ALAD is a polymorphic enzyme showing marked ethnic group differences. In this study, ALAD polymorphism is studied in a Turkish population. Genomic DNA extracted from 230 individuals and polymerase chain reaction (PCR) coupled with the restriction fragment length polymorphism (RFLP) technique were used to identify variants. The frequencies of the alleles ALAD1 and ALAD2 in Turkey were 0.887 and 0.113, respectively. This study provides the first analysis of the allele frequency distribution of the ALAD gene in a Turkish population. The results are compared with other world populations.     

Renal heme metabolism in hereditary tyrosinemia: use of succinylacetone in rat renal tubules.

Succinylacetone (SA), a metabolic end-product found in urine from individuals with hereditary tyrosinemia and associated renal Fanconi syndrome and a known inhibitor of hepatic 5-aminolevulinic acid dehydratase (ALAD), has been used to study heme metabolism in isolated rat renal tubules. Heme biosynthetic porphyrin precursors are increased selectively in the presence of 4 mmol/1 SA. Total porphyrin content of the tubules are increased approximately 2-fold, while both ferrochelatase and heme oxygenase activities remain unaffected by SA. Nonetheless, total heme content is reduced, as was incorporation of radioactive label from amino[14C]levulinic acid. Cytochrome P-450 content remained unaffected. Impairment of iron uptake and/or transport within the cell or enhancement of heme catabolism via a non-heme oxygenase-dependent pathway could explain the observations.