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PubMed2002

Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1.

Dormant spores of the marine Bacillus sp. strain SG-1 catalyze the oxidation of manganese(II), thereby becoming encrusted with insoluble Mn(III,IV) oxides. In this study, it was found that the Mn(II)-oxidizing activity could be removed from SG-1 spores using a French press and recovered in the supernatant following centrifugation of the spores. Transmission electron microscopy of thin sections of SG-1 spores revealed that the ridged outermost layer was removed by passage through the French press, leaving the remainder of the spore intact. Comparative chemical analysis of this layer with the underlying spore coats suggested that this outer layer is chemically distinct from the spore coat. Taken together, these results indicate that this outer layer is an exosporium. Previous genetic analysis of strain SG-1 identified a cluster of genes involved in Mn(II) oxidation, the mnx genes. The product of the most downstream gene in this cluster, MnxG, appears to be a multicopper oxidase and is essential for Mn(II) oxidation. In this study, MnxG was overexpressed in Escherichia coli and used to generate polyclonal antibodies. Western blot analysis demonstrated that MnxG is localized to the exosporium of wild-type spores but is absent in the non-oxidizing spores of transposon mutants within the mnx gene cluster. To our knowledge, Mn(II) oxidation is the first oxidase activity, and MnxG one of the first gene products, ever shown to be associated with an exosporium.

Bacillus↗
PubMed2003

A novel extracellular multicopper oxidase from Phanerochaete chrysosporium with ferroxidase activity.

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2'-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 micro M. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.

Amino Acid Sequence↗
PubMed1999

Tetracopper Assembly Complexes Comprised of One Dimetallic Core and Two Monometallic Auxiliaries: Intramolecular Electron-Transfer Relevant to Multicopper Oxidases.

Two tetracopper assembly complexes, comprised of one dimetallic di(3-iminomethylsalicylato)dicopper(II) core and two monometallic copper(II) auxiliaries attached to the imino nitrogens of the dinuclear core through an alkane chain, have been prepared. [Cu(4)(L(1))](PF(6))(4).2CH(3)CN.3H(2)O (1) has di(2-pyridylmethyl)aminecopper(II) as the monometallic auxiliary, and [Cu(4)(L(2))](ClO(4))(4).CH(3)OH (2) has 1,4,8,11-tetraazacyclotetradecanecopper(II) as the auxiliary. Assembly 1 in acetonitrile shows a two-electron reduction at -0.08 V (vs SCE) followed by a one-electron reduction at -0.42 V. Together with EPR studies for electrolyzed solutions, it is shown that the two monometallic auxiliaries are reduced at -0.08 V, followed by an intramolecular electron transfer from one of the reduced auxiliaries to the dimetallic core and by the second reduction at the resulting monometallic Cu(II) center at -0.42 V: {Cu(II)-Cu(2)(II,II)-Cu(II)}/{Cu(I)-Cu(2)(II,II)-Cu(I)} --> {Cu(I)-Cu(2)(I,II)-Cu(II)}/{Cu(I)-Cu(2)(I,II)-Cu(I)}. The CV of 2 in DMSO shows two couples at -0.68 and -0.99 V attributable to the stepwise reductions: {Cu(II)-Cu(2)(II,II)-Cu(II)}/{Cu(II)-Cu(2)(I,II)-Cu(II)}/{Cu(I)-Cu(2)(I,II)-Cu(II)}. Assembly 1 is reduced with ascorbic acid to the {Cu(I)-Cu(2)(I,II)-Cu(I)} species, whereas 2 is not reduced with ascorbic acid. The relevance of the intramolecular electron transfer observed for 1 to multicopper oxidases is discussed.

Journal Article↗
PubMed1994

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase.

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.

Amino Acid Sequence↗
PubMed2004

Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae.

Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization.

Amino Acid Sequence↗
PubMed2005

Recombinant expression and functional characterization of human hephaestin: a multicopper oxidase with ferroxidase activity.

Human hephaestin (Hp) is a transmembrane protein that has been implicated in duodenal iron export. Mutations in the murine hephaestin gene (sla) produce microcytic, hypochromic anemia that is refractory to oral iron therapy. Hp shares approximately 50% sequence identity with the plasma multicopper ferroxidase ceruloplasmin including conservation of residues involved in disulfide bond formation and metal coordination. On the basis of this similarity to ceruloplasmin, human hephaestin may also bind copper and possess ferroxidase activity. To test this hypothesis, human hephaestin cDNA has been cloned by reverse transcription of human duodenal mRNA. Following in vitro mutagenesis to make the encoded polypeptide suitable for expression and purification, the hephaestin cDNA was cloned into the expression vector pNUT and introduced into baby hamster kidney cells. After selection with methotrexate, the baby hamster kidney cells secreted the recombinant human hephaestin into the medium at a level of approximately 2 mg/L. Purification was achieved by a single immunoaffinity chromatography step. As judged by SDS-PAGE, N-terminal sequence analysis, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, the purified hephaestin was homogeneous with a mass of 129600 Da, suggesting a carbohydrate content of 7.7%. Inductively coupled plasma mass spectrometry revealed that recombinant hephaestin contained an average of 3.13 atoms of copper per protein molecule. A visible absorption maximum was observed at 607 nm, consistent with the presence of a Type 1 copper site. By using ferrous ammonium sulfate as a substrate, recombinant hephaestin was shown to have ferroxidase activity with a K(m) of 2.1 microM for Fe(II). Lastly, urea PAGE showed that hephaestin was able to catalyze formation of diferric transferrin from Fe(II) and apotransferrin.

Animals↗
PubMed1994

The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake.

S. cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter. We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake. The wild-type FET3 gene was isolated by complementation of the mutant defect. Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases. Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport. Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity. We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.

Amino Acid Sequence↗
PubMed2005

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact.

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.

Amino Acid Sequence↗
PubMed2002

Primary structure of a Japanese lacquer tree laccase as a prototype enzyme of multicopper oxidases.

The cDNA library of the Japanese lacquer tree (Rhus vernicifera) was constructed by the reverse transcription of mRNA. A cDNA encoding laccase was amplified by PCR using primers based on the N-terminal amino acid sequences of the purified laccase and its peptide fragments formed by digestions with chymotrypsin and trypsin, and subcloned. The laccase cDNA clone contained a single, large open reading frame of 1599 nucleotides, encoding a protein of 533 amino acids with a calculated molecular mass of 58981 Da. The lacquer laccase was found to have 42 to 62% identity with other plant laccases and 20 to 24% identity with microorganism laccases at the deduced amino acid level. Differing from microorganism laccases the lacquer laccase utilizes a Met residue in addition to one Cys and two His residues to construct the type 1 Cu site. The secondary structure of the lacquer laccase was predicted to mainly consist of the beta-structure (28.7%) and loop and random structures (67.0%). The alpha-helix content was predicted to be only 4.3%. The location of these secondary structures was assumed to be very similar to those of ascorbate oxidase and fungal laccase, the crystal structures of which have been determined.

Amino Acid Sequence↗
PubMed2006

Co-occurrence of the multicopper oxidases tyrosinase and laccase in lichens in sub-order peltigerineae.

BACKGROUND AND AIMS: Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. METHODS: Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. KEY RESULTS: Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. CONCLUSIONS: Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.

Colorimetry↗
PubMed2004

A fungal multicopper oxidase restores iron homeostasis in aceruloplasminemia.

Mutations that lead to a loss of the copper-containing plasma enzyme ceruloplasmin disrupt mammalian iron homeostasis. The mechanism by which ceruloplasmin mobilizes iron from cell stores has been controversial. We demonstrate that injection of a soluble copper-containing yeast protein Fet3p can restore iron homeostasis in phlebotomized mice with a deletion of the ceruloplasmin gene. These results show the conservation of function of copper-containing proteins in eukaryotic iron metabolism.

Animals↗
PubMed2006

Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium.

The US Department of Energy has assembled a high quality draft genome of Phanerochaete chrysosporium, a white rot Basidiomycete capable of completely degrading all major components of plant cell walls including cellulose, hemicellulose and lignin. Hundreds of sequences are predicted to encode extracellular enzymes including an impressive number of oxidative enzymes potentially involved in lignocellulose degradation. Herein, we summarize the number, organization, and expression of genes encoding peroxidases, copper radical oxidases, FAD-dependent oxidases, and multicopper oxidases. Possibly relevant to extracellular oxidative systems are genes involved in posttranslational processes and a large number of hypothetical proteins.

Lignin↗
PubMed1999

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands.

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.

Amino Acid Sequence↗
PubMed2005

The copper-iron connection in biology: structure of the metallo-oxidase Fet3p.

Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O(2) to H(2)O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase.

Ceruloplasmin↗
PubMed1995

Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.

Amino Acid Sequence↗
PubMed1998

Genetic analysis of iron uptake in the yeast Saccharomyces cerevisiae.

OBJECTIVE: We used the methods of yeast genetics to identify genes involved in acquisition of iron by eukaryotic cells. METHODS: Mutants were identified with defects in cellular iron uptake. These were organized into an upstream group and a downstream group. The upstream group was involved in the delivery of copper to the multicopper oxidase FET3. Mutants of this group were characterized by defective iron uptake that could be corrected by exposure of the cells to large amounts of copper. The downstream group was more directly involved in iron uptake. Mutant phenotypes from these genes could not be corrected by copper exposure. RESULTS: Genes in the upstream group encoded the regulator of copper transport, MAC1, and two copper transporters, CTR1 and CCC2. Genes in the downstream group encoded the multicopper oxidase FET3 and its partner the iron permease FTR1. In addition, the downstream genes encoded the surface reductases FRE1 and FRE2 and the iron regulatory protein AFT1. CONCLUSIONS: The iron and copper uptake processes in yeast intersect because the FET3 gene encodes a multicopper oxidase that is required for iron transport. In human beings, an analogous function may be served by ceruloplasmin, a multicopper oxidase with a role in iron homeostasis.

Ceruloplasmin↗
PubMed2005

SKS6, a multicopper oxidase-like gene, participates in cotyledon vascular patterning during Arabidopsis thaliana development.

SKU5-Similar 6 (SKS6) is a one of a large gene family of 19 members in Arabidopsis thaliana (L.) Heynh that encode multicopper oxidase-like proteins that are related to ferroxidases, ascorbate oxidases and laccases. Only one member of the family has been previously studied; Skewed5 (SKU5) is involved in the control of root growth. The encoded SKS6 protein, like SKU5 appears to lack a functional copper-binding site and is most closely related to Bp10 from Brassica napus and Ntp303 from Nicotiana tobacum. The SKS6 promoter contains many putative regulatory sites and differential expression of an SKS6::GUS reporter gene revealed selective induction in several seedling tissues including guard cells, root cortex cells, and leaf margin hydathodes. It was also expressed later in flower development in flower primordia, ovules, and the abscission zones of seeds and siliques. Furthermore, SKS6 was upregulated in roots in response to treatment of seedlings with the hormones abscisic acid, indole-3 acetic acid, 2,4-dichlorophenoxyacetic acid and aminocyclopropane-1-carboxylate. A loss-of function sks6-1 T-DNA insertion allele revealed that cotyledon vascular patterning is affected in the mutant, suggesting a role for the protein in metabolism of nutrients or hormones in the hydathodes, the sites of auxin synthesis and chemical recycling.

Amino Acid Sequence↗