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

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.

Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II).

Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn(2+) to Mn(3+), albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn(2+) turnover (56 s(-1)) nearly in the same order of the two other Mn(2+)-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s(-1) average turnover) and versatile peroxidases (145 s(-1) average turnover), whose genes were also heterologously expressed. Oxidation of Mn(2+) has been reported for an Amycolatopsis DyP (24 s(-1)) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn(2+) oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. It is suggested that generation of Mn(3+) oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn(2+)-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.

Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH.

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn(2+) (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.

A secretomic view of woody and nonwoody lignocellulose degradation by Pleurotus ostreatus.

BACKGROUND: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures. RESULTS: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14-16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10-14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 ß-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends. CONCLUSIONS: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results.

Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding.

Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.

Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment.

BACKGROUND: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases. RESULTS: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of ß-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases. CONCLUSION: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

Homodimeric chicken galectin CG-1B (C-14): Crystal structure and detection of unique redox-dependent shape changes involving inter- and intrasubunit disulfide bridges by gel filtration, ultracentrifugation, site-directed mutagenesis, and peptide mass fingerprinting.

Intrafamily gene diversification has led to three prototype galectins in chicken [i.e., chicken galectin (CG)-1A, CG-1B, and CG-2] that show distinct expression profiles and developmental regulation. In order to pinpoint structural disparities among them, we determined the crystal structure of CG-1B. Alteration of the position of the Trp ring in the lectin site and the presence of only two ordered water molecules therein, as well as changes in the interface region between the two subunits, set the structure of CG-1B clearly apart from that of CG-1A. Intriguingly, the unique presence of two Cys residues at positions 2 and 7 in the N-terminal region translated into formation of an intersubunit disulfide bridge between the Cys7 residues of the homodimer in the crystal. In solution, oxidation is associated with significant shape changes in the dimeric protein and the additional occurrence of a compacted form with an intrasubunit disulfide bridge between Cys2 and Cys7. The single-site mutant C7S/C7V was not subjected to such changes, supporting the crucial role of Cys7 in redox-dependent shape changes. These results point to the functional significance of the distinctive presence of the two Cys residues in the N-terminal region of CG-1B.

Gene design, expression, crystallization and preliminary diffraction analysis of two isolectins from the fungus Coprinus cinereus: a model for studying functional diversification of galectins in the same organism and their evolutionary pathways.

It is the aim of comparative structural biology to define the evolutionarily important traits of protein function and the points of diversification. Consequently, structural analysis, especially of distant members in a family which in this case are lectins involved in cell adhesion and growth regulation in animals (i.e. galectins), is required. For this purpose, recent work has been focused on the first galectins known from outside the animal kingdom. These are the two isolectins from the basidiomycete Coprinus cinereus (inky cap mushroom), termed Cgl-1 and Cgl-2. Additionally, the close similarity (83% deduced amino-acid identity) but the pronounced difference in the expression patterns of these two fungal lectins during fruiting-body formation affords a suitable object for study of the relation of structural difference to the observed functional disparity in the same organism. Both galectins were crystallized after recombinant production. Crystals belong to either the orthorhombic space group C222(1) (Cgl-1) or the monoclinic space group P2(1) (Cgl-2). The latter crystals diffracted to 1.6 A resolution using synchrotron radiation. To solve the phasing problem, a selenomethionine-containing variant of Cgl-1 was designed. Crystals isomorphous to those of the native counterpart were obtained. Their structural analysis will also be crucial to solving the structure of Cgl-2.