5-aminolaevulinic Acid Synthase Research Articles

MDS-516 A Rare Case of X-Linked Recessive Sideroblastic Anemia (XLSA) Masquerading as Myelodysplastic Syndrome-Refractory Anemia With Ringed Sideroblasts (MDS - RARS)

XLSA may be confused clinically with MDS-RARS, which can have patient management implications. Sideroblastic anemia is categorized as a rare type of anemia by National Organization for Rare Disorders. Fewer than 200 cases with less than 100 unrelated probands have been described for X-linked recessive sideroblastic anemia (XLSA). XLSA is the most common type of non-syndromic congenital sideroblastic anemias (CSA), with almost 40% of all CSA cases involving mutations in the ALAS2 (δ- aminolaevulinic acid synthase) gene. Male patients (two-thirds of known cases) usually present in childhood or adolescence with symptoms of anemia, while female patients present in middle age. We present a case of XLSA in a male patient with symptom-onset in middle age. He initially presented with symptoms of anemia and received multiple blood transfusions over a period of 10 years. Subsequent bone marrow biopsy showed a hypercellular bone marrow with erythroid hyperplasia, increased iron and ringed sideroblasts consistent with refractory anemia with ringed sideroblasts, no abnormal karyotype in cytogenetics, and normal flow cytometry results. A liver biopsy showed hemosiderotic changes, and he was initially considered to have myelodysplastic syndrome-refractory anemia with ringed sideroblasts (MDS-RARS). Genetic testing, including SF3B1 gene mutation, revealed no findings suggestive of MDS. The patient was then started on iron chelating agents and pyridoxine with improvement in anemia and did not require any further transfusions with significant improvement in his symptoms. A hereditary anemia NGS gene sequencing and deletion/duplication panel was then done which showed pathogenic X-linked recessive hemizygous mutation involving ALAS2 gene-OMIM 301300. Response to the pyridoxine treatment, absence of SF3B1 gene mutation, and presence of ALAS2 gene mutation helped confirm the diagnosis of XLSA in our patient. This case report highlights the necessity for extensive workup in patients with sideroblastic anemia, given the rarity of XLSA and similarity in its clinical symptoms to MDS-RARS, and the need for its early diagnosis. In our patient, iron overload and subsequent liver cirrhosis developed due to multiple transfusions for refractory sideroblastic anemia which was treated as MDS-RARS and could have been avoided if XLSA had been suspected earlier in the course of the disease.

Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis

Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis

Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis

The first enzyme of haem biosynthesis, ALAS (5-aminolaevulinic acid synthase), catalyses the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolaevulinic acid, CO(2) and CoA. The crystal structure of Rhodobacter capsulatus ALAS provides the first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and structural protein rearrangements, multiple ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-oxoadipate intermediate. Further evidence was obtained by the pH-dependent formation of quinonoid II from the product 5-aminolaevulinic acid. The function of Arg(21), Thr(83), Asn(85) and Ile(86), all involved in the co-ordination of the succinyl-CoA substrate carboxy group, were analysed kinetically. Arg(21), Thr(83)and Ile(86), all of which are located in the second subunit to the intersubunit active site, were found to be essential. Their location in the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization of L-alanine by the ALAS variant T83S indicated the importance of this residue for the selectiveness of binding with the glycine substrate compared with related amino acids. Asn(85) was found to be solely important for succinyl-CoA substrate recognition and selectiveness of binding. The results of the present study provide a novel dynamic view on the structural basis of ALAS substrate-binding and catalysis.

Nitrogen monoxide inhibits haem synthesis in mouse reticulocytes

AI (anaemia of inflammation) often manifests in patients with chronic immune activation due to cancer, chronic infections, autoimmune disorders, rheumatoid arthritis and other diseases. The pathogenesis of AI is complex and involves cytokine-mediated inhibition of erythropoiesis, insufficient erythropoietin production and diminished sensitivity of erythroid progenitors to this hormone, and retention of iron in haemoglobin-processing macrophages. NO (nitric oxide) is a gaseous molecule produced by activated macrophages that has been identified as having numerous effects on iron metabolism. In the present study, we explore the possibility that NO affects iron metabolism in reticulocytes and our results suggest that NO may also contribute to AI. We treated reticulocytes with the NO donor SNP (sodium nitroprusside). The results indicate that NO inhibits haem synthesis dramatically and rapidly at the level of erythroid-specific 5-aminolaevulinic acid synthase 2, which catalyses the first step of haem synthesis in erythroid cells. We also show that NO leads to the inhibition of iron uptake via the Tf (transferrin)-Tf receptor pathway. In addition, NO also causes an increase in eIF2α (eukaryotic initiation factor 2α) phosphorylation levels and decreases globin translation. The profound impairment of haem synthesis, iron uptake and globin translation in reticulocytes by NO raises the possibility that this gas may also contribute to AI.

5-Aminolaevulinic acid synthase and uroporphyrinogen methylase: two key control enzymes of tetrapyrrole biosynthesis and modification.

Two enzymes which play an important role in regulation and flux control through the tetapyrrole biosynthetic pathway are considered. The Rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes are discussed and the progress being made on their recombinant expression and isolation is reported. The Escherichia coli uroporphyrinogen methylase, which is encoded by the cysG gene, is also examined. In this case evidence is provided which demonstrates that the gene product is responsible for the complete synthesis of sirohaem from uroporphyrinogen III. The enzyme is thus capable of performing two S-adenosylmethionine-dependent methylation reactions, an NADP(+)-dependent dehydrogenation and iron chelation. The uroporphyrinogen methylase is thus a small multifunctional enzyme.

Haem repression of the housekeeping 5-aminolaevulinic acid synthase gene in the hepatoma cell line LMH.

Haem is essential for the health and function of nearly all cells. 5-Aminolaevulinic acid synthase-1 (ALAS-1) catalyses the first and rate-controlling step of haem biosynthesis. ALAS-1 is repressed by haem and is induced strongly by lipophilic drugs that also induce CYP (cytochrome P450) proteins. We investigated the effects on the avian ALAS-1 gene promoter of a phenobarbital-like chemical, Glut (glutethimide), and a haem synthesis inhibitor, DHA (4,6-dioxoheptanoic acid), using a reporter gene assay in transiently transfected LMH (Leghorn male hepatoma) hepatoma cells. A 9.1 kb cALAS-1 (chicken ALAS-1) promoter-luciferase-reporter construct, was poorly induced by Glut and not by DHA alone, but was synergistically induced by the combination. In contrast, a 3.5 kb promoter ALAS-1 construct was induced by Glut alone, without any further effect of DHA. In addition, exogenous haem (20 microM) repressed the basal and Glut- and DHA-induced activity of luciferase reporter constructs containing 9.1 and 6.3 kb of ALAS-1 5'-flanking region but not the construct containing the first 3.5 kb of promoter sequence. This effect of haem was subsequently shown to be dependent on the -6.3 to -3.5 kb region of the 5'-flanking region of cALAS-1 and requires the native orientation of the region. Two deletion constructs of this approx. 2.8 kb haem-repressive region (1.7 and 1.1 kb constructs) retained haem-dependent repression of basal and drug inductions, suggesting that more than one cis-acting elements are responsible for this haem-dependent repression of ALAS-1. These results demonstrate that there are regulatory regions in the 5'-flanking region of the cALAS-1 gene that respond to haem and provide a basis for further investigations of the molecular mechanisms by which haem down-regulates expression of the ALAS-1 gene.

Erythroblast Iron Metabolism in Sideroblastic and Sideropenic States

Iron appears to exert self-regulatory control over erythroblast iron uptake, iron storage and its incorporation into haem. It does this via iron regulatory proteins (IRPs) which bind reversibly to the iron responsive elements (IREs) on the mRNA of transferrin receptor (TfR), erythroid 5-aminolaevulinic acid synthase (ALA-S2) and ferritin. Iron deficiency leads to the binding of IRP to IRE. This binding inhibits the translation of mRNA for ALA-S2 and ferritin but stabilizes mRNA for TfR expression.Sideroblastic erythropoiesis is highly ineffective and characterized by mitochondrial iron loading. The study of X-linked sideroblastic anaemia has shown that the entry of iron into the mitochondria is poorly controlled and able to occur when protoporphyrin production is reduced, as is seen with the ALA-S2 mutations, or when it is increased as has been seen with ABC7 transporter mutations.Sideropenia characterises both iron deficiency anaemia (IDA) and the anaemia of chronic disease (ACD). Erythroblasts in ACD seem doubly equipped to protect their iron supply with their ability to increase the efficiency of transferrin-iron uptake as well as to activate the IRP/IRE system to increase surface TfR production. This increase in efficiency restricts the need to increase surface TfR production and maintains serum soluble TfR (sTfR) values within the normal range in iron replete ACD. The coexistence of iron deficiency with chronic disease, however, is associated with an increase in both the efficiency and number and a highly significant rise in sTfR values.

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.

Erythroid gene expression is differentially regulated by erythropoietin, haemin and delta-aminolaevulinic acid in UT-7 cells.

Erythropoietin (Epo) is essential for the later stages of erythropoiesis, acting to promote cell survival and proliferation, but its role in differentiation remains to be defined. The UT-7 cell line exhibits both erythroid and megakaryocytic characteristics and can be induced to differentiate along the erythroid pathway by Epo or the megakaryocytic pathway by phorbol myristic acetate. We have compared the effects of Epo and the chemical inducers, delta-aminolaevulinic acid (delta-ALA) and haemin on the differentiation capacity of UT-7 cells. Epo alone promoted relatively early events in erythroid maturation, without significant changes in haemoglobin production or morphology. GATA-2 and c-myb were down-regulated by Epo, and GATA-2 was further down-modulated by the inducers. Conversely, SCL expression was up-regulated by Epo and further increased by haemin and delta-ALA. Epo caused an increase in the proportion of cells expressing cell surface glycophorin A (GPA) and up-regulated beta- and gamma-globin by several fold. Both haemin and delta-ALA caused a de novo increase in alpha-globin expression as well as enhancing Epo-induced beta-globin expression, leading to a marked increase in haemoglobin production. These results suggest that haemoglobin production in UT-7 cells is limited by a deficiency of erythroid-specific aminolaevulinic acid synthase (ALAS-E) activity or globin synthesis as a consequence of their immaturity as a multipotential cell line.

Liver metabolism of porphyrins and haem.

The liver is an active site for the biosynthesis of haem and porphyrinogens/porphyrins, which are intermediates of the haem biosynthetic pathway, because haem is required for functional activity of the cytochrome P 450 system and other critical hepatic haemoproteins. The production of hepatic haem is regulated primarily through the activity of aminolaevulinic acid synthase which is the first and normally rate-limiting enzyme of the pathway. This is, in turn, controlled by a putative regulatory haem pool. Hepatic haem can be repleted by the intravenous administration of haem, which is the basis for haem therapy in patients with acute porphyric attacks. The liver catabolizes haem to bilirubin through microsomal haem oxygenase activity and excretes haem into bile along with porphyrins. Biliary excretion of porphyrins increases significantly in patients with some types of porphyria. In protoporphyria this may cause liver damage as a result of protoporphyrin toxicity. The delineation of the pathway for protoporphyrin excretion into bile should facilitate therapy in protoporphyria by identifying ways in which protoporphyrin excretion can be enhanced.

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid.

Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.

The effects of bromocriptine and prolactin on porphyrin biosynthesis in the harderian gland of the male hamster, Mesocritecus auratus.

Porphyrin biosynthesis was examined in the Harderian gland of the male golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, 5-aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are low in normal male glands but were greatly raised in males castrated for 6 weeks. However, porphyrin synthesis remained at basal levels in castrates given the dopamine agonist bromocriptine; this suppression could be reversed by simultaneous prolactin administration, and castrated males receiving prolactin alone exhibited very high enzyme activity and porphyrin content. Bromocriptine also prevents the morphological feminisation of the Harderian gland which would normally occur after castration; again, the simultaneous administration of prolactin permits feminisation to occur. The results support the hypothesis that, while androgens have an inhibitory effect on porphyrin synthesis within this model, other factors, including prolactin, are permissive.

Highlights in haem biosynthesis

The haem biosynthesis pathway continues to provide surprises, from the first enzyme, 5-aminolaevulinic acid synthase, the mRNA of which contains an iron-responsive element, to the last, ferrochelatase, that contains an iron sulphur cluster. 5-Aminolaevulinate dehydratases from animals are zinc-dependent enzymes while those from plants require magnesium. The first X-ray structure of a haem synthesis enzyme, porphobilinogen deaminase, has not only yielded clues about the mechanism of tetrapyrrole assembly but has also provided insight into the molecular basis of the human disease acute intermittent porphyria. Evidence is growing to suggest that a previously unsuspected alternative haem pathway may exist.

Betaine Stimulation of Vitamin B12 Biosynthesis in Pseudomonas denitrificans May Be Mediated by an Increase in Activity of -Aminolaevulinic Acid Synthase

The stimulation by betaine of vitamin B12 and extracellular coproporphyrin production in the industrial species Pseudomonas denitrificans GY57/2 was confirmed using a replacement culture technique; biosynthesis of haem was also found to be stimulated. In the absence of betaine, δ-aminolaevulinic acid synthase decreased in specific activity from time zero. In the presence of betaine, the enzyme activity increased markedly after 3 h and then decreased after exhaustion of extracellular betaine. The peak specific activity of δ-aminolaevulinic acid synthase increased in a dose-dependent manner. The enzyme was inhibited by haem. δ-Aminolaevulinic acid dehydratase was present in considerable excess over δ-aminolaevulinic acid synthase under all conditions studied and its specific activity was not affected by the addition of betaine. Our tentative conclusion is that the betaine effect is exerted via some positive effect on δ-aminolaevulinic acid synthase, the initial, rate-limiting and very labile enzyme of porphyrin and corrin biosynthesis.

The synthesis and properties of 4,5-dioxov aleric acid, a possible intermediate in the biosynthesis of 5-aminolaevulinic acid, and its in vivo formation in Scenedesmus obliquus

The formation of 5-aminolaevulinic acid for chlorophyll biosynthesis in the pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus occurs via two pathways: the first and major pathway uses glycine and succinyl-CoA as precursors and the second, the C-5 pathway, uses the intact five C-atom skeleton of either glutamate or 2-oxoglutarate. The intermediates in this latter pathway of 5-aminoalevulinic acid biosynthesis are still a matter of controversy and discussion but, in this paper, we have demonstrated in this Scenedesmus mutant that 4,5-dioxovaleric acid occurs in the cells when illuminated in the presence of laevulinic acid and the amount of 4,5-dioxovaleric acid formed is dependent on the period of illumination. In a preliminary experiment we have shown, under similar conditions, the labelled 4,5-dioxovaleric acid can be obtained from both dl-[1- 14C]glutamate and [2- 14C]glycine. This suggests that 4,5-dioxovaleric acid is formed enzymically from the five C-atom skeleton of glutamate but may also arise from the deamination of ALA formed by the condensation of succinyl-CoA and glycine by the 5-aminolaevulinic acid synthase pathway. To obtain positive identification the naturally occurring, and also the labeled, 4,5-dioxovaleric acid were isolated as the more stable benzoquinoxaline derivative by condensation with 2,3-diaminonaphthalene and identified by comparison with the benzoquinoxaline derivative of an authentic sample of 4,5-dioxovaleric acid prepared by the hydrogenation of ozonide of benzylidene laevulinic acid. The structures of the authentic sample of 4,5-dioxovaleric acid and its benzoquinoxaline derivative were confirmed by NMR and mass spectroscopy and both further characterized by TLC and by infrared, ultraviolet and fluorescence spectroscopy.

Haem biosynthesis in peripheral blood in erythropoietic protoporphyria.

Summary The enzymes of haem biosynthesis have been measured in the peripheral blood of 10 patients with erythropoietic protoporyphria. The activity of leukocyte φ-aminolaevulinic acid synthase was significantly elevated (P < 0.0001) as was that of erythrocyte porphobilinogen deaminase (P = 0′0023). There was a consistent depression of leukocyte ferrochelatase activity (P < 0.0001). This study provides evidence that this disease is related to a generalized genetically determined deficiency of ferrochelatase activity. Control of the pathway is by end product repression and inhibition of φ–aminolaevulinic acid synthase with possible secondary control mechanism at the level of porphobilinogen deaminase.

ENZYME ABNORMALITIES IN THE PORPHYRIAS

Evidence is presented that each of the porphyrias represents a different inborn error of metabolism in hæm biosynthesis. Control of the pathway takes place by feedback repression and inhibition by hæm of &dgr;-aminolaevulinic-acid synthase. It is suggested that in situations where the activity of this enzyme is derepressed, porphobilinogen deaminase represents a secondary control step.