Identification of a mutation in the ovine uroporphyrinogen decarboxylase (UROD) gene associated with a type of porphyria

Porphyria is a group of at least eight diseases caused by abnormalities in the chemical steps that lead to haeme production. The different types of porphyria show different signs and symptoms and can be strongly influenced by environmental factors. Mutations of the uroporphyrinogen decarboxylase (UROD) gene have been shown to be causative for porphyria cutanea tarda (PCT) in humans. Porphyria is a rare disorder in livestock. Although disorders of haeme biosynthesis have been described in cattle, pigs, sheep and cats, PCT has only been reported in pigs. We observed typical signs of porphyria (photosensitivity and porphyrinuria) in a flock of German Blackface sheep and postulated that the porphyria could be caused by a mutation in the UROD gene. To investigate this, we cloned and sequenced the ovine UROD gene. We identified a single point mutation (C --> T) in UROD which leads to an amino acid substitution at Leu 131 Pro, which is located within the active cleft site of the UROD protein.     

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.     

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.     

对生玉米幼叶尿卟啉原脱羧酶的纯化及部分性质研究

尿卟啉原Ⅲ脱羧酶是植物血红素和叶绿素合成的一个关键调控酶。对生玉米叶片中叶绿素含量较比互生玉米高。对生玉米幼苗经硫酸铵分级沉淀,DEAE Sepharose CL-6B、Sephacryl S-200、羟基磷灰石和B lueSepharose CL-6B层析,纯化了尿卟啉原脱羧酶。纯化倍数为1 060倍,得率约8%,比活约880 U/mg蛋白。纯化的UROD在SDS/PAGE显示一条带,亚基分子量约为40 kD,Sephacryl S-300测得全酶分子量约为55 kD。IEF-PAGE显示UROD为一条带,等电点约为6.0,酶的最适pH值约7.0,在55℃下保温12 m in,酶活力丧失90%,在100 mm ol/L的巯基乙醇下,UROD的酶活力提高7倍。体外5 mm ol/L的磷酸吡哆醛修饰显示UROD活力下降约30%。     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.     

Interaction of the Molecular Chaperone HtpG with Uroporphyrinogen Decarboxylase in the Cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

Characterization and crystallization of human uroporphyrinogen decarboxylase.

The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM. URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 a resolution.     

The role of iron in experimental porphyria and porphyria cutanea tarda

Porphyria cutanea tarda (PCT) and experimental porphyria are characterized by a decreased activity of the enzyme uroporphyrinogen decarboxylase, and accumulation of uroporphyrins and heptacarboxylporphyrins in the liver. Iron (Fe) plays an important role in PCT and experimental porphyria. Biochemically and electron microscopically, we examined the relationship between Fe and porphyrins in liver tissue of C57BL/10 mice made porphyric by administration of iron dextran as Imferon^® (IMF), and in liver biopsies of patients with symptomatic PCT. Accumulation of uroporphyrins and heptacarboxylporphyrins, and an increased amount of Fe were observed in livers of mice treated with IMF and in liver biopsies of patients with PCT. In mice treated with IMF, the activity of uroporphyrinogen decarboxylase was decreased. Both in livers of mice treated with IMF and in livers of patients with PCT, needle-like structures, representing uroporphyrin crystals, were observed by electron microscopy. Uroporphyrin crystals and Fe (as ferritin) were observed in the same hepatocyte. Moreover, there was a striking morphological correlation between uroporphyrin crystals and ferritin-Fe, suggesting a role for (ferritin-)Fe in the pathogenesis of 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.Abbreviations ALA 5-aminolevulinic acid - copro coproporphyrin - coprogen coproporphyrinogen - protogen IX protoporphyrinogen IX - UROD uroporphyrinogen decarboxylase - uro uroporphyrin - urogen uroporphyrinogen     

Arginine decarboxylase is a component activity of the multifunctional enzyme putrescine synthase in cucumber seedlings

A homogenous PreParation of Putrescine synthase, the versatile multifunctional enzyme involved in agmatine →Putrescine conversion inCucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the Purified arginine decarboxylase mediated the comPonent as well as the comPlete set of couPled reactions harboured by Putrescine synthase. Both the enzyme PreParations exhibited identical electroPhoretic and chromatograPhic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional Putrescine synthase inCucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterPart inLathyrus sativus.     

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.     

Leishmania spp.: delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.     

Recent advances in heme biocatalysis engineering

Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L⁻¹. This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.     

Upregulation of CYP2e1 and CYP3a activities in histamine-deficient histidine decarboxylase gene targeted mice

Histamine is a biogenic amine with multiple physiological functions. Its importance in allergic inflammation is well characterized; moreover, it plays a role in the regulation of gastric acid production, various hypothalamic functions, such as food uptake, and enhancing TH2 balance during immune responses. Using histidine decarboxylase gene targeted (HDC(-/-)) BALB/c mice, we studied the effect of the absence of histamine on four cytochrome p450 enzyme activities. Their selective substrates were measured: ethoxyresorufin O-dealkylase activity of CYP1A, pentoxyresorufin O-dealkylase activity of CYP2B, chlorzoxazone 6-hydroxylase activity of CYP2E1 and ethylmorphine N-demethylase activity of CYP3A.The results indicate a significant elevation of CYP2E1 and CYP3A activities, however, no change in CYP1A and CYP2B activities was seen in HDC targeted mice compared to wild type controls with identical genetic backgrounds.     

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.     

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.     

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.