Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo. IMPORTANCE: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Identification of a coproporphyrinogen-III oxidase gene and its correlation with nacre color in Hyriopsis cumingii.

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

A Maize Necrotic Leaf Mutant Caused by Defect of Coproporphyrinogen III Oxidase in the Porphyrin Pathway.

Lesion mimic mutants provide ideal genetic materials for elucidating the molecular mechanism of cell death and disease resistance. The maize necrotic leaf mutant (nec-t) is a recessive mutant with necrotic spots and yellow-green leaves. In this study, we found that nec-t was a light and temperature-dependent mutant. Map-based cloning and the allelic test revealed that nec-t was a novel allelic mutant of the Necrotic4 gene. Necrotic4 encodes the coproporphyrinogen III oxidase (CPX1), a key enzyme in the tetrapyrrole pathway, catalyzing coproporphyrinogen III oxidate to protoporphyrinogen IX. Subcellular localization showed that the necrotic4 protein was localized in the chloroplast. Furthermore, RNA-seq analysis showed that the Necrotic4 mutation caused the enhanced chlorophyll degradation and reactive oxygen species (ROS) response. The mechanism of plant lesion formation induced by light and temperature is not clear. Our research provides a basis for understanding the molecular mechanism of necrosis initiation in maize.

Tyrosyl Radical-Mediated Sequential Oxidative Decarboxylation of Coproporphyrinogen III through PCET: Theoretical Insights into the Mechanism of Coproheme Decarboxylase ChdC.

The peroxide-dependent coproheme decarboxylase ChdC from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b, i.e., two sequential oxidative decarboxylations of coproporphyrinogen III (coproheme III) at propionate groups P2 and P4. In the binding site of coproheme III, P2 and P4 are anchored by different residues (Tyr144, Arg217, and Ser222 for P2 and Tyr113, Lys148, and Trp156 for P4); however, strong experimental evidence supports that the generated Tyr144 radical acts as an unique intermediary for hydrogen atom transfer (HAT) from both reactive propionates. So far, the reaction details are still unclear. Herein, we carried out quantum mechanics/molecular mechanics calculations to explore the decarboxylation mechanism of coproheme III. In our calculations, the coproheme Cpd I, Fe(IV) = O coupled to a porphyrin radical cation (por•+) with four propionate groups, was used as a reactant model. Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2. First, the proton-coupled electron transfer (PCET) occurs from Tyr144 to P2, generating a Tyr144 radical, which then abstracts a hydrogen atom from the Cß of P2. The ß-H extraction was calculated to be the rate-limiting step of decarboxylation. It is the porphyrin radical cation (por•+) that makes the PCET from Tyr144 to P2 to be quite easy to initiate the decarboxylation. Finally, the electron transfers from the Cß• through the porphyrin to the iron center, leading to the decarboxylation of P2. Importantly, the decarboxylation of P4 mediated by Lys148 was calculated to be very difficult, which suggests that after the P2 decarboxylation, the generated harderoheme III intermediate should rebind or rotate in the active site so that the propionate P4 occupies the binding site of P2, and Tyr144 again mediates the decarboxylation of P4. Thus, our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.

In order for the light to shine so brightly, the darkness must be present-why do cancers fluoresce with 5-aminolaevulinic acid?

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection and photodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IX phenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies.

Staphylococcus aureus Coproporphyrinogen III Oxidase Is Required for Aerobic and Anaerobic Heme Synthesis.

The virulence of the human pathogen Staphylococcus aureus is supported by many heme-dependent proteins, including key enzymes of cellular respiration. Therefore, synthesis of heme is a critical component of staphylococcal physiology. S. aureus generates heme via the coproporphyrin-dependent pathway, conserved across members of the Firmicutes and Actinobacteria In this work, we genetically investigate the oxidation of coproporphyrinogen to coproporphyrin in this heme synthesis pathway. The coproporphyrinogen III oxidase CgoX has previously been identified as the oxygen-dependent enzyme responsible for this conversion under aerobic conditions. However, because S. aureus uses heme during anaerobic nitrate respiration, we hypothesized that coproporphyrin production is able to proceed in the absence of oxygen. Therefore, we tested the contribution to anaerobic heme synthesis of CgoX and two other proteins previously identified as potential oxygen-independent coproporphyrinogen dehydrogenases, NWMN_1486 and NWMN_1636. We have found that CgoX alone is responsible for aerobic and anaerobic coproporphyrin synthesis from coproporphyrinogen and is required for aerobic and anaerobic heme-dependent growth. This work provides an explanation for how S. aureus heme synthesis proceeds under both aerobic and anaerobic conditions.IMPORTANCE Heme is a critical molecule required for aerobic and anaerobic respiration by organisms across kingdoms. The human pathogen Staphylococcus aureus has served as a model organism for the study of heme synthesis and heme-dependent physiology and, like many species of the phyla Firmicutes and Actinobacteria, generates heme through a coproporphyrin intermediate. A critical step in terminal heme synthesis is the production of coproporphyrin by the CgoX enzyme, which was presumed to be oxygen dependent. However, S. aureus also requires heme during anaerobic growth; therefore, the synthesis of coproporphyrin by an oxygen-independent mechanism is required. Here, we identify CgoX as the enzyme performing the oxygen-dependent and -independent synthesis of coproporphyrin from coproporphyrinogen, resolving a key outstanding question in the coproporphyrin-dependent heme synthesis pathway.

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen†III Oxidase HemN.

HemN is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical-based hydrogen transfer from the propionate to the 5'-deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM-based methylene radical by the vinyl moiety of the mono-decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical-mediated hydrogen transfer in radical SAM enzymology.

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase.

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe2+ or Mg2+ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light-harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light-harvesting polypeptides.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Oxygen-dependent copper toxicity: targets in the chlorophyll biosynthesis pathway identified in the copper efflux ATPase CopA deficient mutant.

Characterization of a copA(-) mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA(-) mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg-chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe-4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.

Erythroid heme biosynthesis and its disorders.

Heme, which is composed of iron and the small organic molecule protoporphyrin, is an essential component of hemoglobin as well as a variety of physiologically important hemoproteins. During erythropoiesis, heme synthesis is induced before, and is essential for, globin synthesis. Although all cells possess the ability to synthesize heme, there are distinct differences between regulation of the pathway in developing erythroid cells and all other types of cells. Disorders that compromise the ability of the developing red cell to synthesize heme can have profound medical implications. The biosynthetic pathway for heme and key regulatory features are reviewed herein, along with specific human genetic disorders that arise from defective heme synthesis such as X-linked sideroblastic anemia and erythropoietic protoporphyria.

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase.

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.

Cerium relieves the inhibition of chlorophyll biosynthesis of maize caused by magnesium deficiency.

Lanthanoids (Ln) were demonstrated to improve chlorophyll formation and the growth of plants. But the mechanism of the fact that Ln promotes chlorophyll biosynthesis of plants is poorly understood. The main aim of the study was to determine Ln effects in chlorophyll formation of maize under magnesium (Mg) deficiency. Maize plants were cultivated in Hoagland's solution. They were subjected to Mg deficiency and to cerium administered in Mg-deficient Hoagland's media, and then the contents of various chlorophyll precursors and gen expressions of the key enzymes of chlorophyll biosynthesis were examined. The decrease of chlorophyll contents in maize leaves caused by Mg deficiency suggested an inhibition of chlorophyll synthesis that was inhibited by a reduction of the precursors as measured by analyzing the contents of δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen III, Mg-protoporphyrin IX, and protochlorophyll, as well as the expression levels of magnesium chelatase, magnesium-protoporphyrin IX methyltransferase, and chlorophyll synthase; Mg deficiency significantly inhibited the transformation from coproporphyrinogen III or protoporphyrin IX to chlorophyll. However, cerium addition significantly relieved the inhibition of chlorophyll biosynthesis in maize caused by Mg deficiency and increased chlorophyll content and promoted a series of transformations from δ-aminolevulinic acid to chlorophyll and maize growth under Mg deficiency. It implied that cerium might partly substitute for the role of Mg.

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.

Cloning, expression, and biochemical properties of CPOX4, a genetic variant of coproporphyrinogen oxidase that affects susceptibility to mercury toxicity in humans.

Coproporphyrinogen oxidase (CPOX) catalyzes the two-step decarboxylation of coproporphyrinogen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg(2+) present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was twofold lower than that of CPOX (CPOX K(m) = 0.30 microM, V(max) = 0.52 pmol protoporphyrin-IX; CPOX4 K(m) = 0.54 microM, V(max) = 0.33 pmol protoporphyrin-IX). Hg(2+) specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40-50%) than those of either wild-type or heterozygous. Additionally, enzymes from mutant, heterozygous and wild-type livers were comparably inhibited by Hg(2+) (10 microM), decreasing CPOX4 activity to 25% that of the wild-type enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.

Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen.

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

Identification of tetrapyrrole compounds excreted by Rhodobacter sphaeroides and sources of the methyl hydrogens of bacteriochlorophyll a biosynthesized by R. sphaeroides, based on 13C-NMR spectral analysis of coproporphyrin III tetramethyl ester.

Red-fluorescent tetrapyrrole compounds excreted by Rhodobacter sphaeroides into the culture broth were concluded to be coproporphyrinogen (Copro'gen) III and uroporphyrinogen (Uro'gen) I, based on the (13)C-NMR spectral identification of coproporphyrin (Copro) III tetramethyl ester and uroproporphyrin (Uro) I octamethyl ester. The sources of the methyl hydrogens of bacteriochlorophyll a were established by analysis of the (13)C-NMR spectra of (2)H,(13)C-Copro III tetramethyl ester chemically derived from (2)H,(13)C-Copro'gen III biosynthesized through the feeding of delta-amino[2-(13)C]levulinic acid (ALA) to R. sphaeroides in medium containing 50% (2)H(2)O. We confirmed the previous finding that one of the methyl hydrogens was derived from water in the medium during decarboxylation of four acetyl side chains of Uro'gen III to generate Copro'gen III. It was further shown that the other hydrogen atoms, previously reported to be derived from methylene hydrogens at C-2 of ALA, had been exchanged with hydrogen of water in the medium in the biosynthetic pathways leading from ALA to Copro'gen III.

Two novel uroporphyrinogen decarboxylase (URO-D) mutations causing hepatoerythropoietic porphyria (HEP).

Hepatoerythropoietic porphyria (HEP) is a rare form of porphyria in humans. The disorder is caused by homozygosity or compound heterozygosity for mutations of the uroporphyrinogen decarboxylase (URO-D) gene. Subnormal URO-D activity results in accumulation of uroporphyrin in the liver, which ultimately mediates the photosensitivity that clinically characterizes HEP. Two previously undescribed URO-D mutations found in a 2-year-old Caucasian boy with HEP, a maternal nonsense mutation (Gln71Stop), and a paternal missense mutation (Gly168Arg) are reported here. Recombinant Gly168Arg URO-D retained 65% of wild-type URO-D activity and studies in Epstein-Barr Virus (EBV)-transformed lymphoblasts indicated that protein levels are reduced, suggesting that the mutant protein might be subjected to accelerated turnover. The crystal structure of Gly168Arg was determined both as the apo-enzyme and with the reaction product bound. These studies revealed little distortion of the active site, but a loop containing residues 167-172 was displaced, possibly indicating small changes in the catalytic geometry or in substrate binding or increased accessibility to a cellular proteolytic pathway. A second pregnancy occurred in this family, and in utero genotyping revealed a fetus heterozygous for the maternal nonsense mutation (URO-D genotype WT/Gln71Stop). A healthy infant was born with no clinical evidence of porphyria.