Secondary Structure in Enzyme-Inspired Polymer Catalysts Impacts Water Oxidation Efficiency.

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Fungal Laccases: Fundamentals, Engineering and Classification Update.

Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and cupredoxin-like folding, along with some common structural determinants. Laccases constitute the largest group of MCOs, with fungal laccases having the greatest biotechnological applicability due to their superior ability to oxidize a wide range of aromatic compounds and lignin, which is enhanced in the presence of redox mediators. The adaptation of these versatile enzymes to specific application processes can be achieved through the directed evolution of the recombinant enzymes. On the other hand, their substrate versatility and the low sequence homology among laccases make their exact classification difficult. Many of the ever-increasing amounts of MCO entries from fungal genomes are automatically (and often wrongly) annotated as laccases. In a recent comparative genomic study of 52 basidiomycete fungi, MCO classification was revised based on their phylogeny. The enzymes clustered according to common structural motifs and theoretical activities, revealing three novel groups of laccase-like enzymes. This review provides an overview of the structure, catalytic activity, and oxidative mechanism of fungal laccases and how their biotechnological potential as biocatalysts in industry can be greatly enhanced by protein engineering. Finally, recent information on newly identified MCOs with laccase-like activity is included.

Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role.

Multicopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure-function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.

Full Copper Resistance in Cupriavidus metallidurans Requires the Interplay of Many Resistance Systems.

The metal-resistant bacterium Cupriavidus metallidurans uses its copper resistance components to survive the synergistic toxicity of copper ions and gold complexes in auriferous soils. The cup, cop, cus, and gig determinants encode as central component the Cu(I)-exporting PIB1-type ATPase CupA, the periplasmic Cu(I)-oxidase CopA, the transenvelope efflux system CusCBA, and the Gig system with unknown function, respectively. The interplay of these systems with each other and with glutathione (GSH) was analyzed. Copper resistance in single and multiple mutants up to the quintuple mutant was characterized in dose-response curves, Live/Dead-staining, and atomic copper and glutathione content of the cells. The regulation of the cus and gig determinants was studied using reporter gene fusions and in case of gig also RT-PCR studies, which verified the operon structure of gigPABT. All five systems contributed to copper resistance in the order of importance: Cup, Cop, Cus, GSH, and Gig. Only Cup was able to increase copper resistance of the Δcop Δcup Δcus Δgig ΔgshA quintuple mutant but the other systems were required to increase copper resistance of the Δcop Δcus Δgig ΔgshA quadruple mutant to the parent level. Removal of the Cop system resulted in a clear decrease of copper resistance in most strain backgrounds. Cus cooperated with and partially substituted Cop. Gig and GSH cooperated with Cop, Cus, and Cup. Copper resistance is thus the result of an interplay of many systems. IMPORTANCE The ability of bacteria to maintain homeostasis of the essential-but-toxic "Janus"-faced element copper is important for their survival in many natural environments but also in case of pathogenic bacteria in their respective host. The most important contributors to copper homeostasis have been identified in the last decades and comprise PIB1-type ATPases, periplasmic copper- and oxygen-dependent copper oxidases, transenvelope efflux systems, and glutathione; however, it is not known how all these players interact. This publication investigates this interplay and describes copper homeostasis as a trait emerging from a network of interacting resistance systems.

Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l.

Multi-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest range of biotechnological applications (particularly those from basidiomycete fungi) due to their ability to oxidise aromatic compounds and lignin. Thus, comprehensive studies for a better classification and structure-function characterisation of MCO families are required. Laccase-ferroxidases (LAC-FOXs) constitute a separate and unexplored group of MCOs with proposed dual features between laccases and ferroxidases. We aim to better define this cluster and the structural determinants underlying putative hybrid activity. We performed a phylogenetic analysis of the LAC-FOXs from basidiomycete fungi, that resulted in two subgroups. This division seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in Fet3p from Saccharomyces cerevisiae. One of these LAC-FOXs (with only one of these residues) from the fungus Heterobasidion annosum s. l. (HaLF) was synthesised, heterologously expressed and characterised to evaluate its catalytic activity. HaLF oxidised typical laccase substrates (phenols, aryl amines and N-heterocycles), but no Fe (II). The enzyme was subjected to site-directed mutagenesis to determine the key residues that confer ferroxidase activity. The mutated HaLF variant with full restoration of the three acidic residues exhibited efficient ferroxidase activity, while it partially retained the wide-range oxidative activity of the native enzyme associated to laccases sensu stricto.

Multicopper oxidases GbAO and GbSKS are involved in the Verticillium dahliae resistance in Gossypium barbadense.

Ascorbate oxidase (AO) and skewed5 (SKU5)-similar (SKS) proteins belong to the multicopper oxidase (MCO) family and play important roles in plants in response to environmental stress via modulation of oxidoreduction homeostasis. Currently, reports on the response of Gossypium barbadense MCO to Verticillium wilt (VW) caused by Verticillium dahliae are still limited. Herein, RNA sequencing of two G. barbadense cultivars of VW-resistant XH21 and VW-susceptible XH7 under V. dahliae treatment, combined with physiological and genetic analysis, was performed to analyze the function and mechanism of multicopper oxidases GbAO and GbSKS involved in V. dahliae resistance. The identified differentially expressed genes are mainly involved in the regulation of oxidoreduction reaction, and extracellular components and signaling. Interestingly, ascorbate oxidase family members were discovered as the most significantly upregulated genes after V. dahliae treatment, including GbAO3A/D, GbSKS3A/D, and GbSKS16A/D. H2O2 and Asc contents, especially reductive Asc in both XH21 and XH7, were shown to be increased. Silenced expression of respective GbAO3A/D, GbSKS3A/D, and GbSKS16A/D in virus-induced gene silencing (VIGS) cotton plants significantly decreased the resistance to V. dahliae, coupled with the reduced contents of pectin and lignin. Our results indicate that AO might be involved in cotton VW resistance via the regulation of cell wall components.

In silico studies disclose the underlying link between binding affinity and redox potential in laccase isoforms.

Laccases are copper-containing enzymes belonging to the family of multicopper oxidases (MCOs). All MCOs use molecular oxygen to oxidize a wide range of organic compounds by radical catalysis. One of the key fundamental properties of laccases is having high or low redox potentials depending on the active site organization. Several experimental studies have been done to rationalize the high and low redox potential laccases (LRPL), however, molecular understanding is still lacking. In this work, we explored the proteomic profile of laccases produced in the fungal cultures, specifically induced with lignocellulosic biomass such as rice straw. This study was undertaken to explain the differences in the high redox and low redox potential values of different laccases using in-silico approaches. Proteomic profiling and structural and sequence analysis revealed a low level of similarity among them. Docking analyses and molecular dynamics simulation analysis revealed that high redox potential laccases (HRPL) are having good binding affinity compared to low or medium redox potential laccases (MRPL). The stability of these complexes was further analyzed based on reactive distances, active site volume comparison and a number of tunnel formations that were observed to be significantly higher for HRPL. Our results indicate that the number of tunnel formations calculated from the simulation's trajectories and available water molecules at the T3 site directly correlates with the laccases' redox potentials. This study will be helpful and provide valuable inputs for the designing of new laccases to improve lignin degradation.Communicated by Ramaswamy H. Sarma.

Crystal structures of a dodecameric multicopper oxidase from Marinithermus hydrothermalis.

Multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.

Using a monocopper-superoxo complex to prepare multicopper-peroxo species relevant to proposed enzyme intermediates.

With the goal of generating a (peroxo)tricopper species analogous to the Peroxy Intermediate proposed for multicopper oxidases, solutions of the copper-superoxide complex [K(Krypt)][LCuO2] (L = N,N'-bis(2,6-diisopropylphenyl)-2,6-pyridinedicarboxamide, Krypt = 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) were reacted with the dicopper(I) complex [(TPBN)Cu2(MeCN)2][PF6]2 at -70 °C (TPBN = N,N,N',N'-tetrakis-(2-pyridylmethyl)-1,4-diaminobutane). A metastable intermediate formed, which on the basis of UV-vis, EPR, and resonance Raman spectroscopy was proposed to derive from reaction of two equivalents of the copper-superoxide with one equivalent of the dicopper(I) complex to yield a complex with two (peroxo)dicopper moieties rather than the desired (peroxo)tricopper PI model. A similar intermediate formed upon reaction of [K(Krypt)][LCuO2] with [(BPMA)Cu(MeCN)][PF6] (BPMA = N,N-bis(2-pyridylmethyl)-methyl-amine), which contained the same donor set as provided by TPBN. Comparison of resonance Raman data and consideration of structural preferences for LCuX species led to hypothesis of a µ-η1:η2-peroxo structure for both intermediates.

Discovery of two novel laccase-like multicopper oxidases from Pleurotus citrinopileatus and their application in phenolic oligomer synthesis.

BACKGROUND: Laccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited. RESULTS: In the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity. CONCLUSIONS: Overall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.

Functional and transcriptomic investigation of laccase activity in the presence of PCB29 identifies two novel enzymes and the multicopper oxidase repertoire of a marine-derived fungus.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs), that can be detected in a variety of environments including the human body, adversely affecting global health. Bioremediation is an emerging field for the detoxification and removal of environmental pollutants, with novel biocatalysts appropriate for this task being in high demand. In this study, a biobank of novel fungal strains isolated as symbionts of marine invertebrates was screened for their ability to remove 2,4,5-trichlorobiphenyl (PCB29). The most efficient strains were studied further for their ability to express laccase activity, the most commonly associated extracellular activity involved in the removal of aromatic pollutants and encoded in fungi by the enzymatic class of multicopper oxidases (MCOs). The strain expressing the highest laccase activity, Cladosporium sp. TM138-S3, was cultivated in the presence of copper ions in a 12 L bioreactor and two enzymes exhibiting laccase activity were isolated from the culture broth through ion-exchange chromatography. The two enzymes, Lac1 and Lac2, were biochemically characterized and showed similar characteristics, although an improved ability to remove PCB29 (up to 71.2%) was observed for Lac2 in the presence of mediators. In parallel, we performed RNAseq of the strain growing in presence and absence of PCB29 and reconstructed its transcriptome assembly. Functional annotation allowed identifying the MCO repertoire of the fungus, consisting of 13 enzymes. Phylogenetic analysis of Ascomycete MCOs further allowed classifying these enzymes, revealing the diversity of laccase activities in Cladosporium sp. TM138-S3.

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System.

RESEARCH BACKGROUND: In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. EXPERIMENTAL APPROACH: We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. RESULTS AND CONCLUSIONS: Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes.

Copper(II) complexes of tripodal ligand scaffold (N3O) as functional models for phenoxazinone synthase.

The copper(II) complexes [Cu(L)NO3] (1-9) of newer N3O ligands (L1-L9) have been synthesized and characterized. The molecular structure of 1, 4, and 7 exhibited nearly a perfect square pyramidal geometry (τ, 0.04-0.11). The Cu-OPhenolate bonds (~ 1.91 Å) are shorter than the Cu-N bonds (~ 2.06 Å) due to the stronger coordination of anionic phenolate oxygen. The Cu(II)/Cu(I) redox potentials of 1-9 appeared around -0.102 to -0.428 V versus Ag/Ag+ in water. The electronic spectra of the complexes showed the d-d transitions around 643-735 nm and axial EPR parameter (g||, 2.243-2.270; A||, 164-179 × 10-4 cm-1) that corresponds to square pyramidal geometry. The bonding parameters α2, 0.760-0.825; ß2, 0.761-0.994; γ2, 0.504-0.856 and K||, 0.698-0.954 and K⊥, 0.383-0.820 calculated from EPR spectra and energies of d-d transitions. The complexes catalyzed the conversion of substrate 2-aminophenol into 2-aminophenoxazine-3-one using molecular oxygen in the water and exhibited the yields of 41-61%. The formation of the product is accomplished by the appearance of a new absorption band at 430 nm and the rates of formation were calculated as 6.98-15.65 × 10-3 s-1 in water. The reaction follows Michaelis-Menten enzymatic reaction kinetics with turnover numbers (kcat) 9.11 × 105 h-1 for 1 and 4.66 × 105 h-1 for 9 in water. The spectral, redox and kinetic studies were performed in water to mimic the enzymatic oxidation reaction conditions.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Multicopper Oxidases in Saccharomyces cerevisiae and Human Pathogenic Fungi.

Multicopper oxidases (MCOs) are produced by microscopic and macroscopic fungal species and are involved in various physiological processes such as morphogenesis, lignin degradation, and defense mechanisms to stress inducing environmental conditions as well as fungal virulence. This review will summarize our current understanding regarding the functions of MCOs present in Saccharomyces cerevisiae and in different human fungal pathogens. Of the two main MCO groups, the first group of MCOs is involved in iron homoeostasis and the second includes laccases. This review will also discuss their role in the pathogenesis of human fungal pathogens.

Manganese (II) removal from aqueous solutions by Cladosporium halotolerans and Hypocrea jecorina.

Manganese (Mn) is toxic at higher concentrations requiring its removal before returning the wastewater to the environment. This article reported the Mn removal of two fungi strains isolated from mine wastewater. ITS rRNA region sequencing identified the fungi strains as Cladosporium halotolerans and Hypocrea jecorina. Mn2+ removal assays were performed in Sabouraud broth with 50 mg L-1 Mn2+ supplemented and bioleaching assays using MnO2 instead of MnSO4 at the same conditions. C. halotolerans removed 96 % of 50 mg L-1 Mn2+ at two weeks without MnO2 bioleaching with 649.9 mg of biomass and H. jecorina removed about 50 % of Mn2+ in 21 days from initial 50 mg of Mn2+ L-1 with 316.8 mg of biomass. Extracellular laccases were present in C. halotolerans agar regardless of the Mn addition. Mn adsorbed was detected on C. halotolerans hyphae. Mn oxidation was positive to H. jecorina by reaction of its medium with Leucoberbelin blue.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

Multicopper oxidases: Biocatalysts in microbial pathogenesis and stress management.

The acquisition of metal ions such as iron, copper and manganese is essential for the survival of microorganisms as these are constituents of metalloproteins including enzymes, storage proteins, structural elements, transcription factors and antimicrobial factors in various biological processes. However, excess of these metal ions is associated with significant toxicity due to spontaneous redox cycling of ions and obstruction of normal metabolic pathways. To overcome this, microbes have developed a variety of metal regulatory systems allowing them to adapt to the changing biotic and abiotic environments. Multi-copper oxidases (MCOs) such as ceruloplasmins, ferroxidases, laccases and nitrite reductases are such regulatory systems employed by microbes to resist the toxicity of metal ions by controlling their oxidation states under aerobic conditions. MCOs help pathogens survive during an infection by evasion of the toxic environment generated by the host immune system and thus are considered necessary determinants of virulence. This review summarizes the role of MCOs in metal homeostasis under stressful conditions and the extent to which these MCOs contribute to microbial virulence within the host that might prove as an esteemed avenue for the development of novel antimicrobial therapies.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.