Bilirubin oxidase from myrothecium verrucaria physically absorbed on graphite electrodes. Insights into the alternative resting from and the sources of activity loss

The oxygen reduction reaction is one of the most important chemical processes in energy converting systems and living organisms. Mediator-less, direct electro-catalytic reduction of oxygen to water was achieved on spectrographite electrodes modified by physical adsorption of bilirubin oxidases from Myrothecium verrucaria. The existence of an alternative resting form of the enzyme is validated. The effect on the catalytic cycle of temperature, pH and the presence of halogens in the buffer was investigated. Previous results on the electrochemistry of bilirubin oxidase and on the impact of the presence of halogens are reviewed and reinterpreted

A differential genome-wide transcriptome analysis : impact of cellular copper on complex biological processes like aging and development

The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development

Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods

We have used combined quantum mechanical and molecular mechanical free-energy perturbation methods in combination with explicit solvent simulations to study the reaction mechanism of the multicopper oxidases, in particular the regeneration of the reduced state from the native intermediate. For 52 putative states of the trinuclear copper cluster, differing in the oxidation states of the copper ions and the protonation states of water- and O2-derived ligands, we have studied redox potentials, acidity constants, isomerisation reactions, as well as water- and O2 binding reactions. Thereby, we can propose a full reaction mechanism of the multicopper oxidases with atomic detail. We also show that the two copper sites in the protein communicate so that redox potentials and acidity constants of one site are affected by up to 0.2 V or 3 pKa units by a change in the oxidation state of the other site

Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

Multicopper ferroxidases catalyze the oxidation of ferrous iron to ferric iron. In yeast and algae, they participate in cellular uptake of iron; in mammals, they facilitate cellular efflux. The mechanisms of iron metabolism in insects are still poorly understood, and insect multicopper ferroxidases have not been identified. In this paper we present evidence that Drosophila melanogaster multicopper oxidase-1 (MCO1) is a functional ferroxidase. We identified candidate iron binding residues in the MCO1 sequence and found that purified recombinant MCO1 oxidizes ferrous iron. An association between MCO1 function and iron homeostasis was confirmed by two observations: RNAi-mediated knockdown of MCO1 resulted in decreased iron accumulation in midguts and whole insects, and weak knockdown increased the longevity of flies fed a toxic concentration of iron. Strong knockdown of MCO1 resulted in pupal lethality, indicating that MCO1 is an essential gene. Immunohistochemistry experiments demonstrated that MCO1 is located on the basal surfaces of the digestive system and Malpighian tubules. We propose that MCO1 oxidizes ferrous iron in the hemolymph and that the resulting ferric iron is bound by transferrin or melanotransferrin leading to iron storage, iron withholding from pathogens, regulation of oxidative stress and/or epithelial maturation. These proposed functions are distinct from those of other known ferroxidases Given that MCO1 orthologs are present in all insect genomes analyzed to date, this discovery is an important step toward understanding iron metabolism in insects

Characterization of multicopper oxidase CopA from Pseudomonas putida KT2440 and Pseudomonas fluorescens Pf-5 : Involvement in bacterial lignin oxidation

CopA is a protein formed as part of a copper resistance operon in Pseudomonas syringae pv tomato, but CopA has also been identified from gene library screening as a potential lignin-oxidising enzyme. Few bacterial homologues for bacterial multi-copper laccases have been identified that can assist in lignin degradation. Bioinformatic analysis revealed that copA and copC genes were found in the genomes of bacterial strains capable of lignin oxidation. In this study, CopA enzymes from bacterial strains with lignin oxidation activity, Pseudomonas putida and P. fluorescens, were heterologously expressed and characterised kinetically, and expression of bacterial CopC proteins was also investigated. Purified CopA enzymes were dependent upon exogenous copper (II) ions for activity when expressed under fully aerated conditions, however after expression under microaerobic conditions with copper reconstitution, the activity was independent of copper addition. The CopA enzymes showed activity towards the laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); syringaldazine (SGZ); guaiacol; 2,6-dimethoxyphenol (DMP) and 2,4-dichlorophenol (DCP). Moreover, CopA proteins were able to oxidise the lignin model compounds guaiacylglycerol-beta-guaiacyl (GGE) and 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), giving oxidised dimerised products; and they were active towards Ca-lignosulfonate, giving vanillic acid as product. A double gene deletion of copA-I and copA-II genes in Pseudomonas putida KT2440 was constructed, and this mutant showed diminished growth capability on different small aromatic compounds related with lignin degradation, when copper salts were present in the media

Fungal ecological strategies reflected in gene transcription - a case study of two litter decomposers.

Microbial communities interplay with their environment through their functional traits that can be a response or an effect on the environment. Here, we explore how a functional trait-the decomposition of organic matter, can be addressed based on genetic markers and how the expression of these markers reflect ecological strategies of two fungal litter decomposer Gymnopus androsaceus and Chalara longipes. We sequenced the genomes of these two fungi, as well as their transcriptomes at different steps of Pinus sylvestris needles decomposition in microcosms. Our results highlighted that if the gene content of the two species could indicate similar potential decomposition abilities, the expression levels of specific gene families belonging to the glycoside hydrolase category reflected contrasting ecological strategies. Actually, C. longipes, the weaker decomposer in this experiment, turned out to have a high content of genes involved in cell wall polysaccharides decomposition but low expression levels, reflecting a versatile ecology compare to the more competitive G. androsaceus with high expression levels of keystone functional genes. Thus, we established that sequential expression of genes coding for different components of the decomposer machinery indicated adaptation to chemical changes in the substrate as decomposition progressed

A multicopper oxidase (Cj1516) and a CopA homologue (Cj1161) are major components of the copper homeostasis system of Campylobacter jejuni

© American Society for Microbiology, 2008. Post-print version of article deposited in accordance with SHERPA RoMEO guidelines.Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter jejuni are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in Escherichia coli. Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal "twin arginine" signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of p-phenylenediamine (p-PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited p-PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in C. jejuni

Identification and characterization of laccase-type multicopper oxidases involved in dye-decolorization by the fungus Leptosphaerulina sp.

13 p.-4 fig.-4 tab.[Background] Fungal laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete laccases have been less thoroughly studied than white-rot basidiomycetous laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools.[Results] A Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds.[Conclusions] The searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.This research was supported by the Program for Interuniversity Cooperation and Scientific Reasearch (PCI) from the Spanish Agency for International Cooperation and Development (AECID), Project AP/033932/11, and the Spanish Project NOESIS BIO2014-56388-R.Peer reviewe