Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer

Ohgari, Yoshiko; Sawamoto, Mari; Yamamoto, Masayoshi; Kohno, Hirao; Taketani, Shigeru

doi:10.1093/hmg/ddi029

Cited by 22 publications

(25 citation statements)

References 23 publications

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“…6C). Given that the FeCH dimer has been described in other organisms (11,24), we suggest that the FeCH oligomer shown on Fig. 6 is a homodimer, the formation of which strictly depends on the presence of the C-terminal extension.…”

Section: Resultsmentioning

confidence: 54%

“…For Saccharomyces cerevisiae and animal FeCHs, the homodimer has been shown to be the active form of the enzyme (11,24). To elucidate the aggregation state of the Synechocystis enzymes, both WT and truncated recombinant FeCHs were purified by affinity chromatography from solubilized membranes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…6), it is likely that dimerization can stabilize FeCH in the Synechocystis cell. For yeast and animal FeCHs, the homodimer is the functional form of the enzyme required for stability (11,24); and from the crystal structure of human FeCH, it is clear that the C-terminal extension promotes dimerization. The plastid FeCH probably also forms a homodimer (35).…”

Section: Discussionmentioning

confidence: 99%

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The C-Terminal Extension of Ferrochelatase Is Critical for Enzyme Activity and for Functioning of the Tetrapyrrole Pathway in Synechocystis Strain PCC 6803

et al. 2008

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Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the ⌬H324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the ⌬H324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the ⌬H324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the ⌬H324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the "extra" flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.

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Section: Resultsmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The C-Terminal Extension of Ferrochelatase Is Critical for Enzyme Activity and for Functioning of the Tetrapyrrole Pathway in Synechocystis Strain PCC 6803

et al. 2008

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“…This led us to the assumption that, in agreement with the situation in mitochondrial FeCH (Grzybowska et al, 2002;Ohgari et al, 2005), the dimer is the active form of the Synechocystis FeCH. Given that the purified DH347-FeCH is fairly active and the same enzyme is fully active in membranes (Fig.…”

Section: The Cab Domain Is Necessary For the Dimerization Of Synechocmentioning

confidence: 69%

Functional Assignments for the Carboxyl-Terminal Domains of the Ferrochelatase from Synechocystis PCC 6803: The CAB Domain Plays a Regulatory Role, and Region II Is Essential for Catalysis

et al. 2010

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Ferrochelatase (FeCH) catalyzes the insertion of Fe2+ into protoporphyrin, forming protoheme. In photosynthetic organisms, FeCH and magnesium chelatase lie at a biosynthetic branch point where partitioning down the heme and chlorophyll (Chl) pathways occurs. Unlike their mammalian, yeast, and other bacterial counterparts, cyanobacterial and algal FeCHs as well as FeCH2 isoform from plants possess a carboxyl-terminal Chl a/b-binding (CAB) domain with a conserved Chl-binding motif. The CAB domain is connected to the FeCH catalytic core by a proline-rich linker sequence (region II). In order to dissect the regulatory, catalytic, and structural roles of the region II and CAB domains, we analyzed a FeCH ƊH347 mutant that retains region II but lacks the CAB domain and compared it with the ƊH324-FeCH mutant that lacks both these domains. We found that the CAB domain is not required for catalytic activity but is essential for dimerization of FeCH; its absence causes aberrant accumulation of Chl-protein complexes under high light accompanied by high levels of the Chl precursor chlorophyllide. Thus, the CAB domain appears to serve mainly a regulatory function, possibly in balancing Chl biosynthesis with the synthesis of cognate apoproteins. Region II is essential for the catalytic function of the plastid-type FeCH enzyme, although the low residual activity of the ƊH324-FeCH is more than sufficient to furnish the cellular demand for heme. We propose that the apparent surplus of FeCH activity in the wild type is critical for cell viability under high light due to a regulatory role of FeCH in the distribution of Chl into apoproteins.

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“…The mammalian enzyme is located in the innermembrane of mitochondria and faces the active site of the matrix (Taketani 1993). The mammalian enzyme contains a 2Fe-2S cluster in the carboxyl terminal region (Wu et al 2001;Ohgari et al 2005). The iron-sulfur cluster is essential for promotion of the high enzyme activity although the cluster is not part of the binding region of the ferrous ion substrate (Furukawa et al 1995;Taketani et al 2000Taketani et al , 2003.…”

Section: Biosynthesis and Regulation Of Heme Metabolism In Mitochondriamentioning

confidence: 99%

Aquisition, Mobilization and Utilization of Cellular Iron and Heme: Endless Findings and Growing Evidence of Tight Regulation

Taketani

2005

Tohoku J. Exp. Med.

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100

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Iron is fastidiously utilized by living cells, since it is an essential element, but is toxic in excess. Cells take up iron via a transferrin-transferrin receptor-dependent endocytotic process. The iron thus taken up is used for essential biological functions including oxygen transport, electron transfer, and DNA synthesis. The intracellular level of iron is tightly controlled, through regulation of the cellular uptake of iron and the sequestering of low molecular labile iron into the storage protein ferritin. The known proteins of iron transport and storage, transferrin, transferrin receptor and ferritin, have been recently linked with a number of newly identified proteins that are responsible for inherited diseases of iron metabolisms and play critical roles in the maintenance of iron homeostasis. These proteins are involved in regulation of intracellular levels of iron, iron transport, and heme transport and the oxygen-dependent regulation of gene expression. On the other hand, most iron is transported into mitochondria and immediately used for the biosynthesis of heme in erythroid cells. The heme biosynthesis in mitochondria is coupled with the supply of iron, and the heme, exported from mitochondria, is utilized as prosthetic groups of hemeproteins. Furthermore, non-erythroid and erythroid cells possess the different regulatory systems for the biosynthesis of heme; iron positively regulates the biosynthesis in erythroid cells while heme negatively regulates it in non-erythroid cells. Because of the toxicity and insolubility of heme, the intracellular level of uncommitted heme is maintained at a low concentration (< 10 -9 M). The influx and efflux of heme also help to prevent cytotoxicity. Finally, heme-binding transcriptional factors such as Bach1 and NPAS2 regulate the transcription of several genes involved in the synthesis and degradation of heme-hemeproteins. The discovery of new molecules related to disorders of iron and heme metabolism is ascribable to a complete mechanistic understanding of the cellular network of iron homeostasis. The network of interactions that link iron and heme metabolisms with functions of cellular regulation involving oxidative stress and inflammations contributes to new insights into clinical aspects of disorders.

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Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer

Cited by 22 publications

References 23 publications

The C-Terminal Extension of Ferrochelatase Is Critical for Enzyme Activity and for Functioning of the Tetrapyrrole Pathway in Synechocystis Strain PCC 6803

The C-Terminal Extension of Ferrochelatase Is Critical for Enzyme Activity and for Functioning of the Tetrapyrrole Pathway in Synechocystis Strain PCC 6803

Functional Assignments for the Carboxyl-Terminal Domains of the Ferrochelatase from Synechocystis PCC 6803: The CAB Domain Plays a Regulatory Role, and Region II Is Essential for Catalysis

Aquisition, Mobilization and Utilization of Cellular Iron and Heme: Endless Findings and Growing Evidence of Tight Regulation

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