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PubMed2005

Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II).

Bacterial oxidation of Mn(II) to Mn(IV) is believed to drive the oxidative segment of the global biogeochemical Mn cycle and regulates the concentration of dissolved Mn(II) in the oceanic water column, where it is a critical nutrient for planktonic primary productivity. Mn(II) oxidizing activity is expressed by numerous phylogenetically diverse bacteria and fungi, suggesting that it plays a fundamental and ubiquitous role in the environment. This important redox system is believed to be driven by an enzyme or enzyme complex involving a multicopper oxidase, although the biochemical mechanism has never been conclusively demonstrated. Here, we show that Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1 is a result of two sequential one-step electron transfer processes, both requiring the putative multicopper oxidase, MnxG, in which Mn(III) is a transient intermediate. A kinetic model of the oxidation pathway is presented, which shows that the Mn(II) to Mn(III) step is the rate-limiting step. Thus, oxidation of Mn(II) appears to involve a unique multicopper oxidase system capable of the overall two-electron oxidation of its substrate. This enzyme system may serve as a source for environmental Mn(III), a strong oxidant and competitor for siderophore-bound Fe(III) in nutrient-limited environments. That metabolically dormant spores catalyze an important biogeochemical process intimately linked to the C, N, Fe, and S cycles requires us to rethink the role of spores in the environment.

Bacillus↗
PubMed2006

Manganese(IV) oxide production by Acremonium sp. strain KR21-2 and extracellular Mn(II) oxidase activity.

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.

Acremonium↗
PubMed1989

Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.

Amino Acid Sequence↗
PubMed2006

A crystallographic and spectroscopic study on the effect of X-ray radiation on the crystal structure of Melanocarpus albomyces laccase.

Laccases (p-diphenol dioxygen oxidoreductases) belong to the family of blue multicopper oxidases, which catalyse the four-electron reduction of dioxygen to water concomitantly through the oxidation of substrate molecules. Blue multicopper oxidases have four coppers, a copper (T1) forming a mononuclear site and a cluster of three coppers (T2, T3, and T3') forming a trinuclear site. Because X-rays are known to liberate electrons during data collection and may thus affect the oxidation state of metals, we have investigated the effect of X-ray radiation upon the crystal structure of a recombinant laccase from Melanocarpus albomyces through the use of crystallography and crystal absorption spectroscopy. Two data sets with different strategies, a low and a high-dose data set, were collected at synchrotron. We have observed earlier that the trinuclear site had an elongated electron density amidst coppers, suggesting dioxygen binding. The low-dose synchrotron structure showed similar elongated electron density, but the high-dose X-ray radiation removed the bulk of this density. Therefore, X-ray radiation could alter the active site of laccase from M. albomyces. Absorption spectra of the crystals (320, 420, and 590nm) during X-ray radiation were measured at a home laboratory. Spectra clearly showed how that the band at 590nm had vanished, resulting from the T1 copper being reduced, during the long X-ray measurements. The crystal colour changed from blue to colourless. Absorptions at 320 and 420nm seemed to be rather permanent. The absorption at 320nm is due to the T3 coppers and it is proposed that absorption at 420nm is due to the T2 copper when dioxygen or a reaction intermediate is close to this copper.

Ascomycota↗
PubMed2001

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system.

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.

Amino Acid Sequence↗
PubMed1986

Isolation and partial nucleotide sequence of the laccase gene from Neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin.

The laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) gene from Neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxyl-terminal region of the protein has been determined. The gene was cloned by cDNA synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(A) RNA isolated from cycloheximide-treated N. crassa cultures as template. Based on the nucleotide sequence of the cDNA obtained, a unique 21-mer was synthesized and used to screen a genomic DNA library from N. crassa. Five different positive clones were isolated and shown to share an overlapping DNA region with the same pattern of restriction sites. Sequence analysis of the common 1.36-kilobase Sal I fragment revealed an open reading frame of 726 nucleotides. The amino acid sequence deduced is in complete agreement with the primary structures of several tryptic peptides isolated previously from N. crassa laccase. The analyzed carboxyl-terminal region of laccase exhibits a striking sequence homology to the carboxyl-terminal part of the third homology unit of the multicopper oxidase ceruloplasmin and to a smaller extent, to the low molecular weight blue copper proteins plastocyanin and azurin. Based on amino acid sequence comparison between these proteins, putative copper ligands of N. crassa laccase are proposed. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene.

Amino Acid Sequence↗
PubMed2006

Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis.

Nitrite represents a bioactive reservoir of nitric oxide (NO) that may modulate vasodilation, respiration and cytoprotection after ischemia-reperfusion injury. Although nitrite formation is thought to occur via reaction of NO with oxygen, this third-order reaction cannot compete kinetically with the reaction of NO with hemoglobin to form nitrate. Indeed, the formation of nitrite from NO in the blood is limited when plasma is substituted with physiological buffers, which suggests that plasma contains metal-based enzymatic pathways for nitrite synthesis. We therefore hypothesized that the multicopper oxidase, ceruloplasmin, could oxidize NO to NO+, with subsequent hydration to nitrite. Accordingly, plasma NO oxidase activity was decreased after ceruloplasmin immunodepletion, in ceruloplasmin knockout mice and in people with congenital aceruloplasminemia. Compared to controls, plasma nitrite concentrations were substantially reduced in ceruloplasmin knockout mice, which were more susceptible to liver infarction after ischemia and reperfusion. The extent of hepatocellular infarction normalized after nitrite repletion. These data suggest new functions for the multicopper oxidases in endocrine NO homeostasis and nitrite synthesis, and they support the hypothesis that physiological concentrations of nitrite contribute to hypoxic signaling and cytoprotection.

Animals↗
PubMed2004

Crystallization and initial X-ray analysis of phenoxazinone synthase from Streptomyces antibioticus.

Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown. As a first step towards determining the three-dimensional structure of the enzyme, phenoxazinone synthase has been crystallized. The hexameric form of phenoxazinone synthase was purified from 72 h cultures of S. lividans containing the plasmid pIJ702. Purified hexamers were concentrated to 75 mg ml(-1) and used to grow two forms of crystals. Data collected from the two crystal forms were processed in two separate space groups. Crystals of both forms were grown at 288 K using the sitting-drop vapour-diffusion method. Native data sets extending to resolutions of 3.35 and 2.30 A have been collected and processed in space groups R32 and P1, respectively.

Crystallization↗
PubMed2006

A ferroxidation/permeation iron uptake system is required for virulence in Ustilago maydis.

In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.

Biological Transport↗
PubMed2003

Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties.

Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.

Amino Acid Sequence↗
PubMed1998

Observation of Cu-N3- stretching and N3- asymmetric stretching bands for mono-azide adduct of Rhus vernicifera laccase.

Mono-azide adduct of Rhus vernicifera laccase, a multicopper oxidase containing one type-1 (blue) copper, one type-2 (non-blue normal) copper, and a pair of type-3 (binuclear and EPR silent) coppers, of which type-2 and type-3 coppers constitute a trinuclear site, was investigated with resonance Raman (RR) and Fourier transform infrared (FT-IR) spectroscopies as a step toward elucidation of the structure and function of the trinuclear site. The Cu-N3- stretching (vCu-N3-) RR band was observed for azide-bound multicopper oxidases for the first time. The vCu-N3- band was located at 400 cm-1 for mono-14N3- laccase, which shifted to 396 cm-1 with the 15N14N14N3- analog. The N3- asymmetric stretching (v(N3-)asym) band was observed by FT-IR spectroscopy at 2035 cm-1 for mono-14N3- laccase and at 2025 cm-1 for the 15N14N14N3- analog. The vCu-N3- and v(N3-)asym frequencies and their 15N14N14N- isotope shifts for azido laccase correspond well with those of metazido hemocyanin, indicating that both derivatives should have a similar binding geometry of azide.

Azides↗
PubMed2002

Enzymatic manganese(II) oxidation by metabolically dormant spores of diverse Bacillus species.

Bacterial spores are renowned for their longevity, ubiquity, and resistance to environmental insults, but virtually nothing is known regarding whether these metabolically dormant structures impact their surrounding chemical environments. In the present study, a number of spore-forming bacteria that produce dormant spores which enzymatically oxidize soluble Mn(II) to insoluble Mn(IV) oxides were isolated from coastal marine sediments. The highly charged and reactive surfaces of biogenic metal oxides dramatically influence the oxidation and sorption of both trace metals and organics in the environment. Prior to this study, the only known Mn(II)-oxidizing sporeformer was the marine Bacillus sp. strain SG-1, an extensively studied bacterium in which Mn(II) oxidation is believed to be catalyzed by a multicopper oxidase, MnxG. Phylogenetic analysis based on 16S rRNA and mnxG sequences obtained from 15 different Mn(II)-oxidizing sporeformers (including SG-1) revealed extensive diversity within the genus Bacillus, with organisms falling into several distinct clusters and lineages. In addition, active Mn(II)-oxidizing proteins of various sizes, as observed in sodium dodecyl sulfate-polyacrylamide electrophoresis gels, were recovered from the outer layers of purified dormant spores of the isolates. These are the first active Mn(II)-oxidizing enzymes identified in spores or gram-positive bacteria. Although extremely resistant to denaturation, the activities of these enzymes were inhibited by azide and o-phenanthroline, consistent with the involvement of multicopper oxidases. Overall, these studies suggest that the commonly held view that bacterial spores are merely inactive structures in the environment should be revised.

Bacillus↗
PubMed2006

Structure of phenoxazinone synthase from Streptomyces antibioticus reveals a new type 2 copper center.

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.

Copper↗
PubMed2002

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Aniline Compounds↗
PubMed2001

Decay of the peroxide intermediate in laccase: reductive cleavage of the O-O bond.

Laccase is a multicopper oxidase that contains four Cu ions, one type 1, one type 2, and a coupled binuclear type 3 Cu pair. The type 2 and type 3 centers form a trinuclear Cu cluster that is the active site for O(2) reduction to H(2)O. To examine the reaction between the type 2/type 3 trinuclear cluster and dioxygen, the type 1 Cu was removed and replaced with Hg(2+), producing the T1Hg derivative. When reduced T1Hg laccase is reacted with dioxygen, a peroxide intermediate (P) is formed. The present study examines the kinetics and mechanism of formation and decay of P in T1HgLc. The formation of P was found to be independent of pH and did not involve a kinetic solvent isotope effect, indicating that no proton is involved in the rate-determining step of formation of P. Alternatively, pH and isotope studies on the decay of P revealed that a proton enhances the rate of decay by 10-fold at low pH. This process shows an inverse k(H)/k(D) kinetic solvent isotope effect and involves protonation of a nearby residue that assists in catalysis, rather than direct protonation of the peroxide. Decay of P also involves a significant oxygen isotope effect (k(16)O(2)/k(18)O(2)) of 1.11 +/- 0.05, indicating that reductive cleavage of the O-O bond is the rate-determining step in the decay of P. The activation energy for this process was found to be approximately 9.0 kcal/mol. The exceptionally slow rate of decay of P is explained by the fact that this process involves a 1e(-) reductive cleavage of the O-O bond and there is a large Franck-Condon barrier associated with this process. Alternatively, the 2e(-) reductive cleavage of the O-O bond has a much larger driving force which minimizes this barrier and accelerates the rate of this reaction by approximately 10(7) in the native enzyme. This large difference in rate for the 2e(-) versus 1e(-) process supports a molecular mechanism for multicopper oxidases in which O(2) is reduced to H(2)O in two 2e(-) steps.

Hydrolysis↗
PubMed2004

Direct electron transfer between copper-containing proteins and electrodes.

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the "blue" multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed.

Adsorption↗
PubMed2025

Decoding the distribution, structure-function-redox potential relationship and recent advances in fungal laccases: a systematic approach.

Laccases, categorized as multicopper oxidases, are recognized for their multifaceted roles in ecosystems and their utility in diverse industrial applications. Laccases from higher fungi, specifically Ascomycota and Basidiomycota, have garnered significant research interest due to their elevated redox potentials and their capacity to degrade lignin in decaying wood, alongside other industrial uses. Here, we have conducted a comprehensive and systematic analysis on fungal laccases using Web of Science, Scopus, PubMed, and ScienceDirect. The genomic distribution, phylogenetic affiliation, and structural organization of laccase-encoding genes in higher fungal species were investigated, as were the catalytic mechanisms of the corresponding enzymes. Additionally, the study explores the correlation between structural domains and redox potential, as well as the impact of post-translational modifications like glycosylation on enzyme activity. Furthermore, the recent advancements in laccase engineering, employing strategies such as rational design, directed evolution, and heterologous expression are discussed. The review also explores the scope of "artificial intelligence and machine learning" in deducing the structure-function relationships, optimizing codon usage, predicting signal peptides, enhancing enzymatic performance, and developing host-specific genetic engineering techniques is also discussed for tailoring fungal laccases to meet the demands of industrial biocatalysis for improved activity and stability.

Laccase↗