Resolving the roles of the Rhodobacter sphaeroides HemA and HemT 5-aminolevulinic acid synthases.

Strains of the phototrophic alpha-proteobacterium Rhodobacter sphaeroides vary in the number of enzymes catalyzing the formation of 5-aminolevulinic acid (ALA synthases) that are encoded in their genomes. All have hemA, but not all have hemT. This study compared transcription of these genes, and also properties of their products among three wild-type strains; 2.4.3 has hemA alone, 2.4.1 and 2.4.9 have both hemA and hemT. Using lacZ reporter plasmids all hemA genes were found to be upregulated under anaerobic conditions, but induction amplitudes differ. hemT is transcriptionally silent in 2.4.1 but actively transcribed in 2.4.9, and strongly upregulated under anaerobic-dark growth conditions when cells are respiring dimethyl sulfoxide, vs. aerobic-dark or phototrophic (anaerobic-light) conditions. Two extracytoplasmic function (ECF)-type sigma factors present in 2.4.9, but absent from 2.4.1 are directly involved in hemT transcription. Kinetic properties of the ALA synthases of all three strains were similar, but HemT enzymes are far less sensitive to feedback inhibition by hemin than HemA enzymes, and HemT is less active under oxidizing conditions. A model is presented that compares and contrast events in strains 2.4.1 and 2.4.9.

[Molecular regulation of heme biosynthesis].

It is generally accepted that the major organs producing heme are erythroid cells and the liver. delta-Aminolevulinate synthase (ALAS) plays the key role to regulate heme biosynthesis in the liver as well as in erythroid cells. In the liver, nonspecific (or housekeeping) isozyme of ALAS (ALAS-N) is expressed to be regulated by its end product, heme, in the negative feedback manner. Not only erythroid isozyme of ALAS (ALAS-E) but also ALAS-N is expressed in erythroid tissues, and are regulated by distinctive manners. For example, heme regulates ALAS-N and ALAS-E in the negative and in the positive feedback manner, respectively. In this article, we describe the molecular mechanisms to regulate heme biosynthesis not only in the liver but also in erythroid cells.

Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes.

The nucleotide sequences of the Rhodobacter sphaeroides hemA and hemT genes, encoding 5-aminolevulinic acid (ALA) synthase isozymes, were determined. ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A, the first and rate-limiting step in tetrapyrrole biosynthesis. The hemA and hemT structural gene sequences were 65% identical to each other, and the deduced HemA and HemT polypeptide sequences were 53% identical, with an additional 16% of aligned amino acids being similar. HemA and HemT were homologous to all characterized ALA synthases, including two human ALA synthase isozymes. In addition, they were evolutionarily related to 7-keto-8-aminopelargonic acid synthetase (BioF) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), enzymes which catalyze similar reactions. Two hemA transcripts were identified, both expressed under photosynthetic conditions at levels approximately three times higher than those found under aerobic conditions. A single transcriptional start point was identified for both transcripts, and a consensus sequence at this location indicated that an Fnr-like protein may be involved in the transcriptional regulation of hemA. Transcription of hemT was not detected in wild-type cells under the physiological growth conditions tested. In a mutant strain in which the hemA gene had been inactivated, however, hemT was expressed. In this mutant, hemT transcripts were characterized by Northern (RNA) hybridization, primer extension, and ribonuclease protection techniques. A small open reading frame of unknown function was identified upstream of, and transcribed in the same direction as, hemA.

5-Aminolevulinate synthase in sideroblastic anemias: mRNA and enzyme activity levels in bone marrow cells.

To examine the role of 5-aminolevulinate synthase (ALAS) in the pathogenesis of sideroblastic anemias, levels of mRNAs for erythroid and housekeeping ALAS isozymes were examined, and total ALAS activity was assessed in bone marrow cells. In two patients with X-linked sideroblastic anemia the levels of mRNA for erythroid ALAS as well as for alpha and beta globin appear to be decreased while levels of mRNA for glycophorin A in both patients were the same as in normal individuals. However, amounts of housekeeping ALAS mRNA were increased two- to threefold in these patients. Total ALAS activity was also increased two- or threefold, perhaps reflecting increased transcription of the housekeeping gene in response to diminished cellular heme in erythroid cells and/or enhanced translation of the erythroid isoform in response to iron accumulation. In a third patient with X-linked sideroblastic anemia ALAS activity was low but increased to twice the normal value after pyridoxine administration, suggesting a structural defect of the enzyme. In a fourth patient, with isolated congenital, pyridoxine-responsive sideroblastic anemia, the erythroid ALAS mRNA was normal and a low enzyme activity was strikingly enhanced by pyridoxal-phosphate albeit to subnormal levels. In idiopathic acquired sideroblastic anemia, ALAS mRNA for both isozymes was normal and enzyme activity was slightly elevated. These observations thus reflect heterogeneous aberrations of erythroid heme synthesis in the various types of sideroblastic anemia and suggest that defects involving erythroid ALAS underlie at least some of them.

Regulation of porphyrin and heme metabolism in the kidney.

Heme biosynthetic capacity within the kidney is localized mainly within the cells of the proximal convoluted tubule. Porphyrin accumulation in response to porphyrinogenic agents occurs predominantly in the cortical nephrons and decreases dramatically in the medullary region. This pattern of heme biosynthetic capacity correlates with the distribution of mixed function oxidase activities in the kidney. The regulation of heme biosynthesis in kidney cells is qualitatively comparable with that observed in liver, but differs with respect to the time required to realize induction of ALA synthetase in response to porphyrinogenic chemicals. This refractoriness may reflect a substantially greater ratio of regulatory or uncommitted heme to overall heme biosynthetic activity in renal cells, as compared with the hepatocyte. Studies on the mechanisms of trace metal-induced renal porphyria support the view that the kidney can play an important, even predominant, role in the etiology of excess urinary porphyrins excreted as a result of disordered porphyrin metabolism. Evidence from clinical studies suggests that the kidney may also play an important role in the etiology and manifestations of inherited and acquired forms of human porphyria.

Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae.

Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency. delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S. cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted. The inhibition of sterol uptake (and growth of sterol auxotrophs) by ALA was dependent on the ability to synthesize heme from ALA. A procedure was developed which allowed selection of strains which would take up exogenous sterols but had no apparent defect in heme or ergosterol biosynthesis. One of these sterol uptake control mutants possessed an allele which allowed phenotypic expression of sterol auxotrophy in a heme-competent background.