Genetic heterogeneity in acute intermittent porphyria: characterisation and frequency of porphobilinogen deaminase mutations in Finland.

The occurrence of different porphobilinogen deaminase mutant types in 68 patients with acute intermittent porphyria from 33 unrelated families in Finland was studied with biochemical and immunological techniques. In this fairly homogenous population four different porphobilinogen deaminase mutant types were identified and their frequencies determined. Most (about 80%) of the mutations were cross reacting immunological material (CRIM) negative, including a large kindred with normal erythrocyte porphobilinogen deaminase activities. The remainder of the families had CRIM positive mutations, including an unusual type (type 2) that had an immunoreactive, non-catalytic porphobilinogen deaminase level considerably greater than the maximal theoretical ratio of CRIM to activity of 2.0 for a single mutant allele. Correlations of the amount of residual porphobilinogen deaminase activity and the occurrence of acute clinical manifestations in each mutant type suggested that CRIM positive type 2 patients may have fewer acute symptoms.     

Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence

The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined.     

Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.     

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

Studies on the porphobilinogen deaminase–uroporphyrinogen cosynthetase system of cultured soya-bean cells

1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. delta-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen.     

Mutants of Escherichia coli K12 accumulating porphobilinogen: a new locus, hemC.

Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described. The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem. Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase. In transduction, the mutation mapped close to genes ilvD and metE at minute 84. The gene was tentatively identified as hemC, coding for porphobilinogen deaminase. The gene symbol hemC replaces the earlier and temporary symbol popE.     

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.     

The three-dimensional structures of mutants of porphobilinogen deaminase: toward an understanding of the structural basis of acute intermittent porphyria.

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.     

Labeling of porphobilinogen deaminase by radioactive 5-aminolevulinic acid in isolated developing pea chloroplasts

A protein had been previously described, which was labeled by radioactive 5-aminolevulinic acid in isolated developing chloroplasts. In the present study we have shown that this protein (Mr approximately equal to 43,000) probably exists as a monomer in the chloroplast stroma. The labeling is blocked if known inhibitors of 5-aminolevulinic acid dehydratase are added to the incubation mixture, and is markedly decreased in intensity if nonradioactive 5-aminolevulinate or porphobilinogen are added to the incubation mixture; other intermediates in the porphyrin biosynthetic pathway, uroporphyrinogen III, uroporphyrin III, and protoporphyrin IX, do not decrease the labeling of the 43-kDa protein appreciably. Nondenaturing gels of the proteins isolated from the incubation with radioactive 5-aminolevulinic acid were stained for porphobilinogen deaminase activity. A series of red fluorescent bands was obtained which coincided with the radioactive bands visualized by autoradiography. It is concluded that the soluble chloroplast protein that is labeled in organello by radioactive 5-aminolevulinic acid is porphobilinogen deaminase.     

Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

Porphyrin and corrinoid mutants of Bacillus subtilis.

Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B12 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.     

Biosynthesis of uroporphyrinogens from porphobilinogen: mechanism and the nature of the process.

The enzymic self-polymerization of prophobilinogen gives rise to the cyclic tetrapyrroles uroporphyrinogen III and uroporphyrinogen I. The former is the precursor of all the natural porphyrins and chlorins. The formation of uroporphyrinogen III is catalysed by a dual enzymic system, porphobilinogen deaminase and uroporphyrinogen III cosynthase. Deaminase polymerizes four porphobilinogen units on the enzymic surface, without liberation of free intermediates into the reaction medium, and forms uroporphyrinogen I. Cosynthase enters into association with the deaminase, and acts as a 'specifier protein' of the latter, changing the mode of porphobilinogen condensation on the enzymic surface. The association is independent of the presence of substrate. While deaminase catalyses the head-to-tail condensation of the porphobilinogen units, the association deaminase-cosynthase catalyses the head-to-head condensation of the same units. As a result different enzyme-bound dipyrrylmethanes are formed form the beginning of the process, and this can be demonstrated by using synthetic dipyrrylmethanes and tripyrranes.     

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.     

Assignment of human porphobilinogen deaminase to 11q24.1----q24.2 by in situ hybridization and gene dosage studies

In situ hybridization and gene dosage-effect studies were conducted to determine the detailed chromosomal location of the gene encoding human porphobilinogen deaminase (PBGD). Red cell PBGD activity was normal in one patient with monosomy for 11q24.2----qter but was increased 1.5 times in another patient with trisomy for 11q22.2----qter. The cDNA probe for PBGD was found to be specifically hybridized to band 11q24. These results suggest that the gene for PBGD is localized within the region 11q24.1----q24.2.     

Biosynthesis of uroporphyrinogens. Interaction among 2-aminomethyltripyrranes and the enzymatic system

Many hypotheses on uroporphyrinogen biosynthesis advanced the possibility that 2-aminomethyltripyrranes formed by porphobilinogen deaminase are further substrates or uroporphyrinogen III co-synthase in the presence of porphobilinogen. These proposals were put to test by employing synthetic 2-aminomethyltripyrranes formally derived from porphobilinogen. None of them was found to be by itself a substrate of deaminase or of co-synthase in the presence of porphobilinogen. The tripyrranes chemically formed uroporphyrinogens by dimerization reactions, and the latter had to be deducted in control runs during the enzymatic studies. Two of the tripyrranes examined, the 2-aminomethyltripyrrane 7 and the 2-aminomethyltripyrrane 8, were found to be incorporated into enzymatically formed uroporphyrinogen III in the presence of porphobilinogen and of the deaminase-co-synthase system. While the former gave only a slight incorporation, the latter was incorporated in about 16%. No incorporation of 8 into uroporphyrinogen I was detected. On the basis of these results, and of the previous results obtained with 2-aminomethyldipyrrylmethanes, an outline of the most likely pathway of uroporphyrinogen III biosynthesis from porphobilinogen is given.     

The heme biosynthetic pathway in the regenerating rat liver. The relation between enzymes of heme synthesis and growth

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one-third and two-third hepatectomized rats increased 1.5-fold and 2.7-fold and the activity of porphobilinogen deaminase increased 2-fold and 4-fold and was inversely correlated with ferrochelatase activity. delta-Aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed. The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.