Functional studies of rat hydroxymethylbilane synthase.

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Hydroxymethylbilane synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. We carried out extensive studies of rat hydroxymethylbilane synthase in the present investigation. The enzymatic reaction rate of the holoenzyme was found to be lower than those of the enzyme-intermediate complexes, which corrected the previous theoretical analysis result. Several mutants were constructed, purified and characterized. D44 was found to play an important role in the disassembly of the enzyme-intermediate complexes. E63 and H78 were important for maintaining the activity of the enzyme at high temperature. Four substrate analogs with variation of porphobilinogen side-chain were synthesized and incubated with the enzyme. Three analogs were found to be weak substrates of the enzyme. All four analogs can be used for the preparation of uroporphyrin I analogs.

Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.

De Novo mutation found in the porphobilinogen deaminase gene in Slovak acute intermittent porphyria patient: molecular biochemical study.

The porphyrias are group of mostly inherited disorders in which a specific spectrum of accumulated and excreted porphyrins and heme precursors are associated with characteristic clinical features. There are eight enzymes involved in the heme synthesis and defects in seven of them cause porphyria. Four of them are described as acute hepatic porphyrias, which share possible precipitation of acute attacks with symptoms engaging the nervous system. Acute intermittent porphyria (AIP), caused by partial deficiency of the porphobilinogen deaminase (PBGD), is the most frequent among hepatic porphyrias. Clinical expression is highly variable and ~ 90 % of AIP heterozygotes remain asymptomatic throughout life. During systematic genetic analysis of the AIP patients diagnosed in the Czech and Slovak Republics, we found a special case of AIP. In a 15-year-old boy with abdominal and subsequent neurological symptomatology, we identified de novo mutation 966insA within the PBGD gene leading to a stop codon after 36 completely different amino acids compared to the wt-sequence. To establish the effects of this mutation on the protein structure, we expressed mutant constructs with described mutation in E. coli and analyzed their biochemical and enzymatic properties. Moreover, computer-assisted protein structure prediction was performed.

Measurement of uroporphyrinogen decarboxylase activity.

Uroporphyrinogen decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.

Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation.

Allosteric inhibition of human lymphoblast and purified porphobilinogen deaminase by protoporphyrinogen and coproporphyrinogen. A possible mechanism for the acute attack of variegate porphyria.

Variegate porphyria (VP) is characterized by photocutaneous lesions and acute neuropsychiatric attacks. Decreased protoporphyrinogen oxidase activity results in accumulation of protoporphyrin (ogen) IX and coproporphyrin (ogen) III. During acute attacks delta-aminolevulinic acid and porphobilinogen also increase, suggesting that porphobilinogen deaminase (PBG-D) may be rate limiting. We have examined the effects of porphyrinogens accumulating in VP on PBG-D activity in Epstein-Barr virus-transformed lymphoblast sonicates from 12 VP and 12 control subjects. Protoporphyrinogen oxidase activity was decreased and protoporphyrin increased in VP lymphoblasts. PBG-D in control lymphoblasts obeyed Michaelis-Menten kinetics (Vmax 28.7 +/- 1.8 pmol/mg per h, Hill coefficient 0.83 +/- 0.07). VP sonicates yielded sigmoidal substrate-velocity curves that did not obey Michaelis-Menten kinetics. Vmax was decreased (21.2 +/- 2.0 pmol/mg per h) and the Hill coefficient was 1.78 +/- 0.17. Addition of protoporphyrinogen IX and coproporphyrinogen III to control sonicates yielded sigmoidal PBG-D substrate-velocity curves and decreased PBG-D Vmax. Addition of porphyrins or uroporphyrinogen III did not affect PBG-D activity. Removal of endogenous porphyrin (ogens) from VP sonicates restored normal PBG-D kinetics. Purified human erythrocyte PBG-D obeyed Michaelis-Menten kinetics (Vmax 249 +/- 36 nmol/mg per h, Km 8.9 +/- 1.5 microM, Hill coefficient 0.93 +/- 0.14). Addition of protoporphyrinogen yielded a sigmoidal curve with decreased Vmax. The Hill coefficient approached 4. These findings provide a rational explanation for the increased delta-aminolevulinic acid and porphobilinogen during acute attacks of VP.

Non-essential activation of rat liver porphobilinogen-deaminase by folic acid.

This report demonstrates the ability of folic acid to activate rat liver porphobilinogen-deaminase (PBG-D). Lineweaver-Burk analysis revealed an increase in Vmax (38%) without affecting the Km. In the concentration range assayed, secondary replots of 1/delta slope and 1/delta intersect versus 1/[folic acid] yielded straight lines, indicating the binding of a single molecule of activator to the enzyme PBG-D, with a KA = 1.66 mM. Results presented here show that folic acid acts as a non-essential activator (alpha = 1; beta = 1.6). The activating effect of folic acid has been observed employing the 35-70% ammonium sulphate precipitated fraction, desalted by dialysis or gel filtration, whereas no action was detected when other partially purified PBG-D preparations were utilized as the enzyme source, suggesting either the presence of sites saturated for the activator, or the existence of a different structural protein conformation, or both.

Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase.

Porphobilinogen deaminase, the polymerase that catalyses the synthesis of preuroporphyrinogen, the linear tetrapyrrole precursor of uroporphyrinogen III, has been crystallized from sodium acetate buffer with polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and the space group is P2(1)2(1)2, with unit cell dimensions a = 88.01 A, b = 75.86 A, c = 50.53 A and alpha = beta = gamma = 90 degrees, indicating a single molecule of 34 kDa in the asymmetric unit. The crystals grow to dimensions of 1 mm x 2 mm x 0.5 mm within two weeks in the dark and are stable in the X-ray beam for at least 40 hours. Diffraction data beyond 1.7 A resolution, observed with a synchrotron radiation source, indicate that a high resolution structure analysis is feasible.

Studies on porphobilinogen-deaminase from Saccharomyces cerevisiae.

Porphobilinogen-deaminase from Saccharomyces cerevisiae has been isolated and partially purified 80- and 230-fold in the absence or presence of phenylmethylsulphonyl fluoride, respectively. Some properties of the isolated enzyme were studied. Porphyrin formation was linear with time and protein concentration. Optimum pH was about 7.5-7.8. Molecular mass of the protein was 30,000 +/- 3000 Dalton when the enzyme was purified in the presence of phenylmethylsulphonyl fluoride. A less active and unstable 20,000 Da molecular mass species was obtained when purification was performed in the absence of the protease inhibitor. Porphobilinogen-deaminase exhibited classical Michaelis-Menten kinetics. The apparent Km for uroporphyrinogen formation was 19 microM; Vmax was 3.6 nmol uroporphyrin/h and the Hill coefficient was n = 1. Also the action of several reagents on the activity was studied. Protective thiol agents had no effect. Heavy metals inhibited both porphyrin formation and porphobilinogen consumption, but known sulphydryl inactivating chemicals inhibit the former without modifying the latter. Ammonium ions had no effect on the activity while hydroxylamine completely inhibited both porphyrin formation and porphobilinogen consumption.

Studies on uroporphyrinogen biosynthesis in pig liver.

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Vmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

Purification of human erythrocyte porphobilinogen deaminase.

Human erythrocyte porphobilinogen deaminase was isolated using ammonium sulphate fractionation and heat treatment, Sephadex G-25 and G-100 chromatography, di-ethylamino-ethyl anion-exchange chromatography, chromatofocusing over a pH gradient of 7-4 and, finally, hydrophobic interaction chromatography on a phenyl-Sepharose column. The enzyme appeared pure as judged by sodium-dodecylsulphate-polyacrylamide gel electrophoresis with silver staining, and yielded a 7 115-fold purification.

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.

Porphobilinogen deaminase in human erythrocytes: purification of two forms with apparent molecular weights of 40 kDa and 42 kDa.

Porphobilinogen deaminase was purified from human erythrocytes by ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. Two forms of the enzyme were isolated, with apparent molecular weights of 40 kDa and 42 kDa, and in relative amounts of 85% and 15%, respectively. Both forms were found to have an N-terminal amino acid sequence identical to that published for the erythropoietic form of porphobilinogen deaminase, as deduced from a cDNA clone. The two forms present could each be separated into three differently charged subforms by Mono Q chromatography.

The mouse porphobilinogen deaminase gene. Structural organization, sequence, and transcriptional analysis.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

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.