Harderoporphyria due to homozygosity for coproporphyrinogen oxidase missense mutation H327R.

Hereditary coproporphyria (HCP) is an autosomal dominant acute hepatic porphyria due to the half-normal activity of the heme biosynthetic enzyme, coproporphyrinogen oxidase (CPOX). The enzyme catalyzes the step-wise oxidative decarboxylation of the heme precursor, coproporphyrinogen III, to protoporphyrinogen IX via a tricarboxylic intermediate, harderoporphyrinogen. In autosomal dominant HCP, the deficient enzymatic activity results primarily in the accumulation of coproporphyrin III. To date, only a few homozygous HCP patients have been described, most having Harderoporphyria, a rare variant due to specific CPOX mutations that alter enzyme residues D400-K404, most patients described to date having at least one K404E allele. Here, we describe a Turkish male infant, the product of a consanguineous union, who presented with the Harderoporphyria phenotype including neonatal hyperbilirubinemia, hemolytic anemia, hepatosplenomegaly, and skin lesions when exposed to UV light. He was homoallelic for the CPOX missense mutation, c.980A>G (p.H327R), and had massively increased urinary uroporphyrins I and III (9,250 and 2,910 µM, respectively) and coproporphyrins I and III (895 and 19,400 µM, respectively). The patient expired at 5 months of age from an apparent acute neurologic porphyric attack. Structural studies predicted that p.H327R interacts with residue W399 in the CPOX active site, thereby accounting for the Harderoporphyria phenotype.

The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX.

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.

Normal and abnormal heme biosynthesis. 6. Synthesis and metabolism of a series of monovinylporphyrinogens related to harderoporphyrinogen. Further insights into the oxidative decarboxylation of porphyrinogen substrates by coproporphyrinogen oxidase.

A series of vinylporphyrinogens were prepared to probe the enzyme coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,c-biladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral substituents, was also a substrate for CPO. Furthermore, a nonnatural type I isomer of harderoporphyrinogen was shown to be acted on by CPO, but in this case further metabolism was noted and this afforded an unprecedented trivinyl porphyrinogen product. The corresponding porphyrin methyl ester was isolated and characterized by FAB MS and proton NMR spectroscopy. The results from these studies allow the binding requirements of CPO to be further assessed and provide a series of substrates to investigate this poorly understood enzyme.

Simple formation of an abiotic porphyrinogen in aqueous solution.

Porphyrins have long been proposed as key ingredients in the emergence of life yet plausible routes for forming their essential pyrrole precursor have heretofore not been identified. Here we show that the anaerobic reaction of δ-aminolevulinic acid (ALA, 1-5 mM) with the ß-ketoester methyl 4-methoxyacetoacetate (2-40 mM) in water (pH5-7) at 70-100°C for >6 h affords the porphyrinogen, which upon chemical oxidation gives the corresponding porphyrin in overall yield of up to 10%. The key intermediate is the α-methoxymethyl-substituted pyrrole, which undergoes tetramerization and macrocycle formation under kinetic control. The resulting type-I porphyrin bears four propionic acid and four carbomethoxy groups, is distinct from porphyrins (e.g., uroporphyrin or coproporphyrin) derivable from ALA alone via the extant universal biosynthetic path to tetrapyrroles, and is photoactive upon assembly into cationic micelles in aqueous solution. The simple self-organization of eight acyclic molecules into a tetrapyrrole macrocycle, from which a porphyrin is derived that is photoactive in lipid assemblies, augurs well for the spontaneous origin of catalysts and pigments essential for prebiotic metabolism and proto-photosynthesis.

The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase.

Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.

Harderoporphyria: a variant hereditary coproporphyria.

Three siblings with intense jaundice and hemolytic anemia at birth were found to excrete a high level of coproporphyrin in their urine and feces; the pattern of fecal porphyrin excretion was atypical for hereditary coproporphyria because the major porphyrin was harderoporphyrin (greater than 60%; normal value is less than 20%). The lymphocyte coproporphyrinogen III oxidase activity of each patient was 10% of control values, which suggests a homozygous state. Both parents showed only mild abnormalities in porphyrin excretion and lymphocyte coproporphyrinogen III oxidase activity decreased to 50% of normal values, as is expected in heterozygous cases of hereditary coproporphyria. Kinetic parameters of coproporphyrinogen III oxidase from these patients were clearly modified, with a Michaelis constant 15-20-fold higher than normal values when using coproporphyrinogen or harderoporphyrinogen as substrates. Maximal velocity was half the normal value, and we also observed a marked sensitivity to thermal denaturation. The possibility that a mutation affecting the enzyme on the active center which is specifically involved in the second decarboxylation (from harderoporphyrinogen to protoporphyrinogen) was eliminated by experiments on rat liver that showed that coproporphyrinogen and harderoporphyrinogen were metabolized at the same active center. The pattern of porphyrin excretion and the coproporphyrinogen oxidase from the three patients exhibited abnormalities that were different from the abnormalities found in another recently described homozygous case of hereditary coproporphyria. We suggest naming this variant of coproporphyrinogen oxidase defect "harderoporphyria."

Effects of iron-EDTA on uroporphyrinogen oxidation by liver microsomes.

Uroporphyrinogen oxidation by hepatic microsomes from chick embryos or mice pretreated with methylcholanthrene was increased by addition of iron-EDTA. This increase was partially prevented by catalase, mannitol, ketoconazole and piperonyl butoxide, whereas only ketoconazole and piperonyl butoxide inhibited the oxidation in the presence and absence of iron-EDTA. These data suggest that the oxidations of uroporphyrinogen in the presence and absence of added iron occur by different mechanisms.

Kinetic studies of novel di- and tri-propionate substrates for the chicken red blood cell enzyme coproporphyrinogen oxidase.

Coproporphyrinogen oxidase is an important enzyme in heme biosynthesis and catalyses the sequential oxidative decarboxylation of propionates on the A and B rings of the porphyrinogen ring. The effects of substituents on the C and D rings have not been systematically evaluated for their effects on the kinetic constants, K(m) and V(max). A series of synthetic porphyrinogens have been tested for their ability to affect these kinetic constants for the chicken enzyme. The enzyme exhibited the largest V(max) when incubated with the authentic substrate and was clearly able to distinguish between various substituents on the C and D rings of the macrocycle. When co-incubated with substrate, the authentic product, protoporphyrinogen-IX, appears to inhibit coproporphyrinogen oxidase and this may have an important role in the regulation of this enzyme. Thus the model for the active site of this enzyme should be modified to take these factors into account.

Erythrocyte porphyrinogen carboxy-lyase activity in porphyria cutanea tarda and certain other human porphyrias.

Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria. The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females. In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p less than 0.05 level. Patients with variegata porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity. The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed. The significance of the results with respect to the chemical picture of different porphyrias is discussed.

Porphyria. Basic science aspects.

The porphyrias are a heterogeneous group of clinical disorders that share a common etiologic background in that each manifests a major metabolic defect in the synthesis of heme. The porphyrias represent an important category of human disease, perhaps as much for what they can teach us about how metabolic abnormalities are translated into clinical manifestations as they are as diseases per se. In this discussion an effort is made to describe, in some detail, the metabolic steps involved in the synthesis of heme and to correlate known abnormalities in this sequence of reactions that are associated with human porphyria.

Comparison between crab hepatopancreas and rat liver uroporphyrinogen decarboxylase.

We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the crab Chasmagnathus granulatus as a first step to establish this enzyme as a possible biomarker for environmental contamination. We performed a comparative study of crab UroD with the enzyme UroD present in Wistar rat liver, which is known as a useful indicator of intoxication by polyhalogenated aromatic hydrocarbons (PAHs). The final products were the same in crab and rat UroD: the remaining substrate (8-carboxyl-porphyrinogen), the final product Coproporphyrinogen (4-COOH) and intermediate compounds with 7-, 6- and 5-COOH. The elimination of the second carboxyl group seems to be the rate-limiting step in this multiple decarboxylation, because large amounts of 7-COOH porphyrinogen are accumulated. The V(max)/K(m) ratio was 100-fold higher for rat liver UroD than for crab hepatopancreas UroD, suggesting a higher efficiency of the rat enzyme. Optimum pH for enzyme activity was 7.2 and 6.8 for crab and rat, respectively. Although both systems showed the same optimum temperature (47 degrees C), the activation energy was clearly different, 51.5 kJ/mol for C. granulatus and 5.4 kJ/mol for Rattus norvegicus (Wistar strain). Superdex 75 gel chromatography yielded a single symmetrical peak with an apparent molecular mass of 48+/-3 kDa for crab hepatopancreas UroD, suggesting the existence of only one enzymatic species in C. granulatus.

The Escherichia coli protein YfeX functions as a porphyrinogen oxidase, not a heme dechelatase.

UNLABELLED: The protein YfeX from Escherichia coli has been proposed to be essential for the process of iron removal from heme by carrying out a dechelation of heme without cleavage of the porphyrin macrocycle. Since this proposed reaction is unique and would represent the first instance of the biological dechelation of heme, we undertook to characterize YfeX. Our data reveal that YfeX effectively decolorizes the dyes alizarin red and Cibacron blue F3GA and has peroxidase activity with pyrogallal but not guiacol. YfeX oxidizes protoporphyrinogen to protoporphyrin in vitro. However, we were unable to detect any dechelation of heme to free porphyrin with purified YfeX or in cellular extracts of E. coli overexpressing YfeX. Additionally, Vibrio fischeri, an organism that can utilize heme as an iron source when grown under iron limitation, is able to grow with heme as the sole source of iron when its YfeX homolog is absent. Plasmid-driven expression of YfeX in V. fischeri grown with heme did not result in accumulation of protoporphyrin. We propose that YfeX is a typical dye-decolorizing peroxidase (or DyP) and not a dechelatase. The protoporphyrin reported to accumulate when YfeX is overexpressed in E. coli likely arises from the intracellular oxidation of endogenously synthesized protoporphyrinogen and not from dechelation of exogenously supplied heme. Bioinformatic analysis of bacterial YfeX homologs does not identify any connection with iron acquisition but does suggest links to anaerobic-growth-related respiratory pathways. Additionally, some genes encoding homologs of YfeX have tight association with genes encoding a bacterial cytoplasmic encapsulating protein. IMPORTANCE: Acquisition of iron from the host during infection is a limiting factor for growth and survival of pathogens. Host heme is the major source of iron in infections, and pathogenic bacteria have evolved complex mechanisms to acquire heme and abstract the iron from heme. Recently Létoffé et al. (Proc. Natl. Acad. Sci. U.S.A. 106:11719-11724, 2009) reported that the protein YfeX from E. coli is able to dechelate heme to remove iron and leave an intact tetrapyrrole. This is totally unlike any other described biological system for iron removal from heme and, thus, would represent a dramatically new feature with potentially profound implications for our understanding of bacterial pathogenesis. Given that this reaction has no precedent in biological systems, we characterized YfeX and a related protein. Our data clearly demonstrate that YfeX is not a dechelatase as reported but is a peroxidase that oxidizes endogenous porphyrinogens to porphyrins.