HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae.
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HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription. (+info)
Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two bound S-adenosyl-L-methionines.
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The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S](1+) cluster in two slightly different conformations. Mossbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S](2+) cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of delta = 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a delta = 0.57 (3) mm/s, which shifted to a delta = 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mutants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features. (+info)
Mutations in human CPO gene predict clinical expression of either hepatic hereditary coproporphyria or erythropoietic harderoporphyria.
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Hereditary coproporphyria (HCP), an autosomal dominant acute hepatic porphyria, results from mutations in the gene that encodes coproporphyrinogen III oxidase (CPO). HCP (heterozygous or rarely homozygous) patients present with an acute neurovisceral crisis, sometimes associated with skin lesions. Four patients (two families) have been reported with a clinically distinct variant form of HCP. In such patients, the presence of a specific mutation (K404E) on both alleles or associated with a null allele, produces a unifying syndrome in which hematological disorders predominate: 'harderoporphyria'. Here, we report the fifth case (from a third family) with harderoporphyria. In addition, we show that harderoporphyric patients exhibit iron overload secondary to dyserythropoiesis. To investigate the molecular basis of this peculiar phenotype, we first studied the secondary structure of the human CPO by a predictive method, the hydrophobic cluster analysis (HCA) which allowed us to focus on a region of the enzyme. We then expressed mutant enzymes for each amino acid of the region of interest, as well as all missense mutations reported so far in HCP patients and evaluated the amount of harderoporphyrin in each mutant. Our results strongly suggest that only a few missense mutations, restricted to five amino acids encoded by exon 6, may accumulate significant amounts of harderoporphyrin: D400-K404. Moreover, all other type of mutations or missense mutations mapped elsewhere throughout the CPO gene, lead to coproporphyrin accumulation and subsequently typical HCP. Our findings, reinforced by recent crystallographic results of yeast CPO, shed new light on the genetic predisposition to HCP. It represents a first monogenic metabolic disorder where clinical expression of overt disease is dependent upon the location and type of mutation, resulting either in acute hepatic or in erythropoietic porphyria. (+info)
Structural basis of hereditary coproporphyria.
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Hereditary coproporphyria is an autosomal dominant disorder resulting from the half-normal activity of coproporphyrinogen oxidase (CPO), a mitochondrial enzyme catalyzing the antepenultimate step in heme biosynthesis. The mechanism by which CPO catalyzes oxidative decarboxylation, in an extraordinary metal- and cofactor-independent manner, is poorly understood. Here, we report the crystal structure of human CPO at 1.58-A resolution. The structure reveals a previously uncharacterized tertiary topology comprising an unusually flat seven-stranded beta-sheet sandwiched by alpha-helices. In the biologically active dimer (K(D) = 5 x 10(-7) M), one monomer rotates relative to the second by approximately 40 degrees to create an intersubunit interface in close proximity to two independent enzymatic sites. The unexpected finding of citrate at the active site allows us to assign Ser-244, His-258, Asn-260, Arg-262, Asp-282, and Arg-332 as residues mediating substrate recognition and decarboxylation. We favor a mechanism in which oxygen serves as the immediate electron acceptor, and a substrate radical or a carbanion with substantial radical character participates in catalysis. Although several mutations in the CPO gene have been described, the molecular basis for how these alterations diminish enzyme activity is unknown. We show that deletion of residues (392-418) encoded by exon six disrupts dimerization. Conversely, harderoporphyria-causing K404E mutation precludes a type I beta-turn from retaining the substrate for the second decarboxylation cycle. Together, these findings resolve several questions regarding CPO catalysis and provide insights into hereditary coproporphyria. (+info)
Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells.
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Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process. (+info)
Analysis of hemF gene function and expression in Rhodobacter sphaeroides 2.4.1.
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The hemF gene of Rhodobacter sphaeroides 2.4.1 is predicted to code for an oxygen-dependent coproporphyrinogen III oxidase. We found that a HemF- mutant strain is unable to grow under aerobic conditions. We also determined that hemF expression is controlled by oxygen, which is mediated, at least in part, by the response regulatory protein PrrA. (+info)
The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN.
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During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4. (+info)
Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells.
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Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l(-1), 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers. (+info)