Uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) catalyzes the decarboxylation of uroporphyrinogen III to coproporphyrinogen III. The amino acid sequences, kinetic properties, and physicochemical characteristics of enzymes from different sources (mammals, yeast, bacteria) are similar, but little is known about the structure/function relationships of uroporphyrinogen decarboxylases. Halogenated and other aromatic hydrocarbons cause hepatic uroporphyria by decreasing hepatic uroporphyrinogen decarboxylase activity. Two related human porphyrias, porphyria cutanea tarda and hepatoerythropoietic porphyria, also result from deficiency of this enzyme. The roles of inherited and acquired factors, including iron, in the pathogenesis of human and experimental uroporphyrias are reviewed.     

Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

Cell-free translation of human uroporphyrinogen decarboxylase mRNAs

Uroporphyrinogen decarboxylase was synthesized in a reticulocyte lysate cell-free system under the direction of messenger RNAs isolated from human fetal liver and from human reticulocytes. The enzyme was specifically isolated by immuno affinity chromatography. Analysis of the translation products showed that uroporphyrinogen decarboxylase was synthesized in vitro with its mature molecular weight. This enzyme represented 0.04% of the total neosynthesized proteins under the direction of fetal liver mRNA and about ten times less (0.005%) with reticulocyte mRNA.     

Immunoreactive uroporphyrinogen decarboxylase is unchanged in porphyria caused by TCDD and hexachlorobenzene

A method has been developed for the immuno-titration of rodent liver uroporphyrinogen decarboxylase (porphyrinogen carboxy-lyase, EC 4.1.1.37) and used to show that two porphyrogenic polyhalogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin and hexachlorobenzene, cause porphyria in rodents by decreasing the catalytic activity of uroporphyrinogen decarboxylase without altering the amount of immunoreactive enzyme protein. Investigation of the nature of the inactive form of uroporphyrinogen decarboxylase produced by these compounds should provide new information about the mechanism of their toxicity.     

An effective chromatographic separation of chicken red blood cell coproporphyrinogen oxidase and uroporphyrinogen decarboxylase, two enzymes in heme biosynthesis

Of the heme biosynthetic pathway enzymes, coproporphyrinogen oxidase is one of the least understood. Substrate recognition studies [Prepr. Biochem. Biotech.1997, 27, 47, J. Org. Chem.1999, 64, 464] have been done using chicken blood hemolysates (CBH) as the source of this enzyme. However, the enzyme uroporphyrinogen decarboxylase is also present in these preparations and separation of these two enzymes from CBH had not yet been achieved. Thus, a substrate ligand column was developed by covalently linking coproporphyrin-III to a sepharose resin following a similar procedure previously used for the purification of uroporphyrinogen decarboxylase [Int. J. Biochem.1992, 24, 105]. The ligand-resin chromatography step rapidly separates coproporphyrinogen oxidase from uroporphyrinogen decarboxylase as well as the majority of the hemoglobin.     

Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z

1. A 423-fold purified fraction of uroporphyrinogen decarboxylase (EC 4.1.1.37) showing a specific activity of 770 units/mg protein has been employed in order to study some properties in etiolated Euglena gracilis Z. 2. Uroporphyrinogen decarboxylase has a relative molecular mass of 54,000, an optimum pH of 7.2 and exhibits Michaelis-Menten kinetics, employing both uroporphyrinogen I and uroporphyrinogen III as substrates. 3. Anaerobic conditions seem not to be necessary for uroporphyrinogen decarboxylase activity. Neither EDTA nor cysteine affected enzyme activity, whereas dithiothreitol produced a remarkable activation of coproporphyrinogen formation. 4. Kinetic data employing both substrates showed an accumulation of porphyrinogen (i.e. hexa- and hepta-porphyrin) containing six or seven COOH groups, depending on the uroporphyrinogen concentration used. 5. An unusual elution profile of the intermediates on Sephacryl S-200 was found.     

Assignment of uroporphyrinogen decarboxylase (UROD) to the pter----p21 region of human chromosome 1

The assignment of the human gene for uroporphyrinogen decarboxylase (UROD) to chromosome 1 is confirmed and further localized to the pter----p21 region through the use of human-mouse somatic cell hybrids. Human and mouse UROD were separated by electrophoresis and identified with antibodies to the human enzyme after electrophoretic transfer to nitrocellulose membranes.     

Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.     

Evidence for two uroporphyrinogen decarboxylase isoenzymes in human erythrocytes

In animals and plants, uroporphyrinogen decarboxylase catalyzes the stepwise decarboxylations of uroporphyrinogen, the precursor of heme and chlorophyll. To better understand its metabolic roles, we characterized the enzyme purified to electrophoretic homogeneity (about 11,000-fold) from human erythrocytes by a novel uroporphyrin-sepharose affinity chromatographic method. Native polyacrylamide disc gel electrophoresis of the purified enzyme preparation showed two bands detected by staining either for protein or with uroporphyrin-I. Each individual protein eluted from the gel when subjected to re-electrophoresis on SDS-polyacrylamide gel, appeared as a single protein band with molecular masses of approximately 54,000 and approximately 35,000 daltons respectively. Both proteins were able to catalyze all four decarboxylation steps, though the ratios of enzyme activity using octa-, hepta-, hexa- to pentacarboxylic porphyrinogen substrates were distinctly different. Also, their kinetic analysis with heptacarboxylic porphyrinogen-I substrate provided distinctly different apparent Michaelis constants. This provides the first evidence that decarboxylations of uroporphyrinogen to coproporphyrinogen are catalyzed by two isoenzymes.     

Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Enzymatic and immunological studies of uroporphyrinogen decarboxylase in familial porphyria cutanea tarda and hepatoerythropoietic porphyria

Uroporphyrinogen decarboxylase activity was measured in hemoglobin-free lysates from two patients with hepatoerythropoietic porphyria (HEP) and from 12 unrelated patients with familial porphyria cutanea tarda (PCT). In HEP patients, enzyme activities were 5% of normal, and familial studies clearly confirmed that patients with HEP are cases of homozygous PCT. Immunoreactive uroporphyrinogen decarboxylase was measured by developing a direct and noncompetitive enzyme immunoassay (EIA). For the 12 familial PCT patients, we found an immunoreactive protein decreased (51%) to the same extent as the catalytic activity (48%) [cross-reactive immunological material ( CRIM ) negative]. The children from the HEP family were also CRIM negative, contrasting with another HEP family previously described as CRIM positive; our data support the hypothesis of a heterogeneity in familial uroporphyrinogen decarboxylase deficiency.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

Defective human erythrocyte uroporphyrinogen decarboxylase in familial porphyria cutanea tarda: the metabolic lesion or the result of endogenous porphyrinemia?

We have demonstrated that oral charcoal therapy is as effective as therapeutic phlebotomy in reducing porphyrinemia in porphyria cutanea tarda. The effects of immediate and sustained reduction of porphyrinemia on the catalytic properties of partially purified (approximately 200-fold) preparations of red cell uroporphyrinogen decarboxylase of a patient with familial porphyria cutanea tarda were studied. All populations of the patient's red cells exhibited defective enzyme activity, and the apparent Michaelis constants (Km) determined with penta-, hepta-, and octa-carboxylic I porphyrinogen substrates were approximately 3-4 times higher as compared to the normal controls. Mixing experiments (normal and defective enzyme), and preincubation of the normal enzyme with porphyric plasma prior to purification, yielded data supporting the concept that the catalytic defects of red cell uroporphyrinogen decarboxylase in familial porphyria cutanea tarda are independent of interactions between circulating endogenous porphyrins and the enzyme.     

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker''s yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.     

Random decarboxylation of uroporphyrinogen III by human hepatic uroporphyrinogen decarboxylase

The type III heptacarboxylic porphyrinogens derived from enzymic decarboxylation of an acetic acid substituent on uroporphyrinogen III to a methyl group by human hepatic uroporphyrinogen decarboxylase has been analysed by reversed-phase high-performance liquid chromatography with electrochemical detection. The results showed that all four possible heptacarboxylic acid porphyrinogen isomers, with the methyl group attached to rings A, B, C and D of the tetrapyrrole macrocycle, respectively, were formed in almost equal proportions. It was concluded that the normal pathway of uroporphyrinogen III decarboxylation in human liver follows a random mechanism.     

Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.