Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human uroporphyrinogen decarboxylase.

A recombinant human uroporphyrinogen decarboxylase (E.C. 4.1.1.37, UROD) has been expressed in Escherichia coli and purified to homogeneity. Crystals grew by the hanging-drop vapor-diffusion technique from a starting solution containing 1.5 mg ml-1 protein. The crystals belong to the trigonal space group P3121 or its enantiomer P3221 and diffract to 3 A resolution. The unit-cell parameters are a = b = 103.4, c = 75.7 A and gamma = 120 degrees. The asymmetric unit contains one molecule. Preliminary structural predictions suggest for the protein a TIM-barrel type tertiary structure.

A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.

The maize lesion mimic gene Les22 is defined by dominant mutations and characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner. Phenotypically, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. We have cloned Les22 by using a Mutator-tagging technique. It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and Les22 along with our observation that Les22 is also associated with an accumulation of uroporphyrin revealed what appears to be a case of natural porphyria in plants.

Mouse uroporphyrinogen decarboxylase: cDNA cloning, expression, and mapping.

Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria. The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library. The longest clone of 1.5 kb, designated pmUROD-1, had 5' and 3' untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da. The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli. In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1. The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria.

Isolation, sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley.

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination. Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (233 nmol/kg) causes a significant increase of hepatic uroporphyrin, heptacarboxyporphyrin, and total porphyrins in female C57BL/6 mice, ovariectomized C57BL/6 mice, male C57BL/10 mice, and male C57BL/6 mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 mumol/kg. Cotreatment of the mice with TCDD (233 mol/kg) plus MCDF (750 mumol/kg) resulted in partial antagonism of TCDD-induced hepatic porphyrin accumulation only in the female mice. Parallel studies in female C57BL/6 mice showed that the TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the depression of uroporphyrinogen decarboxylase (UROD). MCDF (750 mumol/kg) did not significantly affect these enzymes. In the cotreatment studies (MCDF plus TCDD), MCDF partially antagonized TCDD-induced hepatic porphyrin accumulation but did not affect the levels of hepatic AHH, EROD, or UROD. These results indicate that other factors, in addition to the induction of cytochrome P450-dependent monooxygenases and depressed UROD activity, are important in TCDD-induced porphyria in C57BL/6 female mice.

Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis.

Three genes hemE, hemF, hemG taking part in the porphyrin biosynthesis of Baccillus subtilis were mapped by two- and three-factor transduction crosses. The gene hemE determines uroporphyrinogen decarboxylase (EC 4.1.1.37) the gene hemF coproporphyrinogen oxidase (EC 1.3.3.3) and the gene hemG ferrochelatase (EC 4.99.1.1) enzymes. The loci hemE, hemF, hemG, are not linked to hemA locus and located near the argC and metD loci.