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PubMed2003

Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae.

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+. Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ produced by Fet3p is a ligand for the iron permease, Ftr1p. Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type. Deletion of FTR1 leads to copper sensitivity also. Production in the ftr1delta strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity. This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1delta strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+. In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta strain; these genes encode the two major copper transporters in laboratory yeast strains. This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper. These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast. Its functional homologs, e.g. ceruloplasmin and hephaestin, could play a similar role in mammals.

Cell Membrane↗
PubMed2001

Intestinal metal ion absorption: an update.

Recent progress in the field of metal ion transport has significantly advanced our understanding of the mechanisms of intestinal metal ion absorption under normal and pathological conditions. In this brief review, we focus on the key proteins involved in intestinal absorption of iron, zinc, and copper. Following the initial description of the apical iron transporter, DCT1, the basolateral transporter complex has been identified, which consists of the metal transporter IREG1/MTP1 and the multicopper oxidase, hephaestin. Novel zinc and copper transporters have been identified as well, mostly based on their homology to yeast and plants transporters. The identification of a variety of copper and zinc transporters is consistent with the importance of copper and zinc in a wide variety of enzymatic reactions, free radical scavenging, and transcriptional control.

Journal Article↗
PubMed1996

Molecular biology of iron acquisition in Saccharomyces cerevisiae.

In recent years, significant advances have been made in our understanding of the mechanism and regulation of elemental iron transport in the eukaryote Saccharomyces cerevisiae. This organism employs two distinct iron-transport systems, depending on the bioavailability of the metal. In iron-replete environments, a low-affinity transport system (K(m) = 30 microM) is used to acquire iron. This system may also be used to acquire other metals including cobalt and cadmium. When environmental iron is limiting, a high-affinity (K(m) = 0.15 microM) iron-transport system is induced. Genetic studies in S. cerevisiae have identified multiple genes involved in both iron-transport systems. Cell-surface reductases, FRE1 and FRE2, provide ferrous iron for both systems. A non-ATP-dependent transmembrane transporter (FET4) has been identified as the main component of low-affinity transport. One gene identified to date as part of the high-affinity transport system is FET3, which shows high sequence and functional homology to multicopper oxidases. Accessory genes required for the functioning of this transport system include a plasma-membrane copper transporter (CTR1), an intracellular copper transporter (CCC2), and a putative transcription factor (AFT1). The mechanism by which these genes act in concert to ensure iron accumulation in S. cerevisiae presents an intriguing picture, drawing parallels with observations made in the human system almost 40 years ago.

Biological Transport↗
PubMed2005

Transcriptional response of Escherichia coli to external copper.

Transcriptional response of Escherichia coli upon exposure to external copper was studied using DNA microarray and in vivo and in vitro transcription assays. Transcription of three hitherto-identified copper-responsive genes, copA (copper efflux transporter), cueO (multicopper oxidase) and cusC (tripartite copper pump component) became maximum at 5 min after addition of copper sulphate, and thereafter decreased to the preshift levels within 30 min. Microarray analysis at 5 min after addition of copper indicated that a total of at least 29 genes including these three known genes were markedly and specifically affected (28 upregulated and one downregulated). Transcription of the divergent operons, cusCFB and cusRS, was found to be activated by CusR, which bound to a CusR box between the cusC and cusR promoters. Except for this site, the CusR box was not identified in the entire E. coli genome. On the other hand, transcription of copA and cueO was found to be activated by another copper-responsive factor CueR, which bound to a conserved inverted repeat sequence, CueR box. A total of 197 CueR boxes were identified on the E. coli genome, including the CueR box associated with the moa operon for molybdenum cofactor synthesis. At least 10 copper-induced genes were found to be under the control of CpxAR two-component system, indicating that copper is one of the signals for activation of the CpxAR system. In addition, transcription of yedWV, a putative two-component system, was activated by copper in CusR-dependent manner. Taken together we conclude that the copper-responsive genes are organized into a hierarchy of the regulation network, forming at least four regulons, i.e. CueR, CusR, CpxR and YedW regulons. These copper-responsive regulons appear to sense and respond to different concentrations of external copper.

Base Sequence↗
PubMed2002

The sctR of Salmonella enterica serova Typhimurium encoding a homologue of MerR protein is involved in the copper-responsive regulation of cuiD.

We have identified the cuiD gene in Salmonella enterica serova Typhimurium that codes for a putative multicopper oxidase. Expression of cuiD is induced by copper ion and its promoter/operator has sequence similarity to the promoters controlled by the transcriptional regulators of the MerR family. We also identified and isolated a gene from S. enterica serova Typhimurium that encodes a 138-amino acid residue protein, sctR, a new member of the MerR family of transcriptional regulators. Transposon-insertional disruption of sctR shows sensitivity to copper ion and no response of cuiD expression. Copper-responsive induction and copper tolerance were restored by providing sctR in trans, suggesting that SctR plays an important role in intracellular copper detoxification.

Amino Acid Sequence↗
PubMed2025

Integrative spatial transcriptomic analysis pinpoints the role of the ferroxidase, TaMCO3, in wheat root tip iron mobilization.

Roots play a critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about the molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences result in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (-Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilization. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple multicopper oxidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Further characterization of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the -Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to -Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in -Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

Triticum↗
PubMed1996

A trinuclear intermediate in the copper-mediated reduction of O2: four electrons from three coppers.

The reaction of metal complexes with dioxygen (O2) generally proceeds in 1:1, 21, or 41 (metal:O2) stoichiometry. A discrete, structurally characterized 31 product is presented. This mixed-valence trinuclear copper cluster, which contains copper in the highly oxidized trivalent oxidation state, exhibits O2 bond scission and intriguing structural, spectroscopic, and redox properties. The relevance of this synthetic complex to the reduction of O2 at the trinuclear active sites of multicopper oxidases is discussed.

Copper↗
PubMed2005

Regulation of resistance to copper in Xanthomonas axonopodis pv. vesicatoria.

Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the cop and pco operons of Pseudomonas syringae and Escherichia coli, respectively. However, instead of the two-component (copRS and pcoRS) systems determining copper-inducible expression of the operons in P. syringae and E. coli, a novel open reading frame, copL, was found to be required for copper-inducible expression of the downstream multicopper oxidase copA in X. axonopodis. copL encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within copL, as well as an amino acid substitution generated at the putative start codon of copL, caused a loss of copper-inducible transcriptional activation of copA. A nonpolar insertion of a kanamycin resistance gene in copL resulted in copper sensitivity in the wild-type strain. However, repeated attempts to complement copL mutations in trans failed. Analysis of the genomic sequence databases shows that there are copL homologs upstream of copAB genes in X. axonopodis pv. citri, X. campestris pv. campestris, and Xylella fastidiosa. The cloned promoter area upstream of copA in X. axonopodis pv. vesicatoria did not function in Pseudomonas syringae or in E. coli, nor did the P. syringae cop promoter function in Xanthomonas. However, a transcriptional fusion of the Xanthomonas cop promoter with the Pseudomonas copABCDRS was able to confer resistance to copper in Xanthomonas, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria.

Bacterial Proteins↗
PubMed2006

Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes.

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.

Bacillus↗
PubMed1996

Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1.

The marine Bacillus sp. strain SG-1 forms spores that oxidize manganese(II) as a result of the activities of uncharacterized components of its spore coat. Nucleotide sequence analysis of chromosomal loci previously identified through insertion mutagenesis as being involved in manganese oxidation identified seven possible genes (designated mnxA to mnxG) in what appears to be an operon. A potential recognition site for the sporulation, mother-cell-specific, RNA polymerase sigma factor, sigmaK, was located just upstream of the cluster, and correspondingly, measurement of beta-galactosidase activity from a Tn917-lacZ insertion in mnxD showed expression at mid-sporulation to late sporulation (approximately stage IV to V of sporulation). Spores of nonoxidizing mutants appeared unaffected with respect to their temperature and chemical resistance properties and germination characteristics. However, transmission electron microscopy revealed alterations in the outermost spore coat. This suggests that products of these genes may be involved in the deposition of the spore coat structure and/or are spore coat proteins themselves. Regions of the deduced protein product of mnxG showed amino acid sequence similarity to the family of multicopper oxidases, a diverse group of proteins that use multiple copper ions to oxidize a variety of substrates. Similar regions included those that are involved in binding of copper, and the addition of copper at a low concentration was found to enhance manganese oxidation by the spores. This suggests that the product of this gene may function like a copper oxidase and that it may be directly responsible for the oxidation of manganese by the spores.

Amino Acid Sequence↗
PubMed1999

A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus.

Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.

Amino Acid Sequence↗
PubMed2001

CotA of Bacillus subtilis is a copper-dependent laccase.

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.

Bacillus subtilis↗
PubMed2001

Genes involved in copper homeostasis in Escherichia coli.

Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E. coli.

Bacterial Proteins↗
PubMed2002

Ceruloplasmin metabolism and function.

Ceruloplasmin is a serum ferroxidase that contains greater than 95% of the copper found in plasma. This protein is a member of the multicopper oxidase family, an evolutionarily conserved group of proteins that utilize copper to couple substrate oxidation with the four-electron reduction of oxygen to water. Despite the need for copper in ceruloplasmin function, this protein plays no essential role in the transport or metabolism of this metal. Aceruloplasminemia is a neurodegenerative disease resulting from inherited loss-of-function mutations in the ceruloplasmin gene. Characterization of this disorder revealed a critical physiological role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores and has provided new insights into human iron metabolism and nutrition.

Ceruloplasmin↗
PubMed2002

Anemia and iron overload due to compound heterozygosity for novel ceruloplasmin mutations.

Aceruloplasminemia is a recessive disorder characterized by anemia, iron overload, and neurodegeneration, caused by the absence of ceruloplasmin (Cp), a multicopper oxidase important for iron export. Few patients homozygous for loss of function mutations of the Cp gene have been reported. We describe a 62-year-old white woman with heavy liver iron overload, diabetes, anemia, and neurologic symptoms. She was compound heterozygote for 2 novel mutations that result in the absence of hepatocyte Cp: an adenine insertion at nucleotide 2917 causing a truncated protein and a C-G transversion causing a glutamine-->glutamic acid substitution at position 146. Although rare in whites, aceruloplasminemia should be considered in the differential diagnosis of unexplained anemia associated with iron overload, because these features anticipate progressive neurologic symptoms. We propose that anemia, secondary to the impaired macrophage iron release, plays a major role in hepatic iron overload through increased absorption mediated by the erythroid regulator.

Anemia↗
PubMed2006

Comparative genomics of regulation of heavy metal resistance in Eubacteria.

BACKGROUND: Heavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification. RESULTS: We studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases. CONCLUSION: Our data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity.

Bacteria↗
PubMed2004

Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis.

In 1987, Miyajima et al. first characterized an autosomal recessive, adult-onset neurodegenerative disorder resembling Parkinson's disease associated with near-absent circulating serum ceruloplasmin levels. Coined "familial apoceruloplasmin deficiency", they described a patient with a presenting triad of diabetes mellitus, retinal degeneration, and neurodegeneration with blepharospasm. Neuropathological evaluation revealed abundant iron deposition in selected neurons of the basal ganglia and substantia nigra with associated neuronal dropout and spongioform degeneration without evidence of reactive gliosis. Subsequently, mutations in the ceruloplasmin gene have been determined to result in the excessive iron accumulation seen in the pancreas, retina, and brain. Elevated serum ferritin suggests a systemic iron overload syndrome, yet affected patients had low transferrin saturation and a mild anemia. This new disease, "aceruloplasminemia", reveals a role for ceruloplasmin as an essential ferroxidase critical for iron homeostasis. This multicopper oxidase promotes efficient iron efflux such that individuals lacking ceruloplasmin develop a presumed oxidative injury secondary to iron accumulation and significant neuronal damage. Aceruloplasminemic mice provide a valuable model to further study the mechanisms by which ceruloplasmin regulates iron trafficking and the role of iron in oxidative injury. Despite the dependence of ceruloplasmin on copper for its function, aceruloplasminemia represents an iron storage disease and not a defect in copper metabolism. However, recent evidence in Saccharomyces cerevisiae indicates that Fet3, the yeast homologue of ceruloplasmin, functions as an essential cuprous oxidase. Further investigation into the mechanisms by which ceruloplasmin regulates iron and copper homeostasis will provide valuable insight into the pathogenesis of metallo-mediated diseases and elucidate mechanisms for transition metal (copper, iron) neuropathology.

Animals↗