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Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi
Abstract
Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.
... The genomes of all 3 species are available on GenBank [accession numbers LXTA00000000 (Stagonospora sp.), LXSZ00000000 (Pyrenochaeta sp.), and LXPO00000000 (P. sporulosum)], and detailed analyses of their secretome composition have been published previously (Zeiner et al., 2016(Zeiner et al., , 2017. ...
Manganese (Mn) oxides are among the strongest oxidants and sorbents in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and manganese peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Here we use a combination of chemical and in-gel assays and bulk mass spectrometry to demonstrate secretome-based Mn(II) oxidation in three phylogenetically diverse Ascomycetes that is mechanistically distinct from hyphal-associated Mn(II) oxidation on solid substrates. We show that Mn(II) oxidative capacity of these fungi is dictated by species-specific secreted enzymes and varies with secretome age, and we reveal the presence of both Cu-based and FAD-based Mn(II) oxidation mechanisms in all 3 species, demonstrating mechanistic redundancy. Specifically, we identify candidate Mn(II)-oxidizing enzymes as tyrosinase and glyoxal oxidase in Stagonospora sp. SRC1lsM3a, bilirubin oxidase in Stagonospora sp. and Paraconiothyrium sporulosum AP3s5-JAC2a, and GMC oxidoreductase in all 3 species, including Pyrenochaeta sp. DS3sAY3a. The diversity of the candidate Mn(II)-oxidizing enzymes identified in this study suggests that the ability of fungal secretomes to oxidize Mn(II) may be more widespread than previously thought.
... Modifying the initial mass of peptides for separation altered organism specific coverage for the soil metaproteome Although the fractionation of the two different peptide masses did not appear to significantly alter the overall protein counts for both metaproteome samples, we evaluated the impact on the proteomes of individual organisms within the larger metaproteome. For the soil, proteins were statistically associated with organisms using BLAST and the UniProt Proteomes Database (Zeiner et al., 2016(Zeiner et al., , 2017The UniProt, 2017) then grouped into individual proteomes. Approximately 470 organisms were associated with proteins identified from the 100 μg initial peptide mass, while 437 organisms were associated with proteins from the 25 μg initial peptide mass. ...
Metaproteomics conducted on soil is challenged by a low depth of protein coverage that can potentially result in an underrepresentation of the functional underpinnings of important biological processes and interactions. Typically, the utilization of an on-line two-dimensional chromatographic separation approach (2D LC-MS/MS) can significantly improve depth of coverage. Herein, we evaluate different fractionation modalities to determine the optimal approach for LC MS based soil metaproteomics. The first approach fractionates the digested soil proteome in 2 dimensions while coupled directly to the MS instrument ("online" approach). The second approach performs the first dimension of fractionation "offline" prior to injection to the MS ("offline" approach). While both approaches are commonly utilized for proteomic research, they have not been directly compared for soils. We rigorously compared these approaches applied to: 1) a mock community consisting of 47 different microorganisms, and 2) to natural soil. The results provide insight into protein dynamic range, the presence of mass spectrometry interfering substances, and other factors that may contribute to an observed low metapro-teome depth of coverage from complex and highly diverse samples, such as soil. We observed that the "offline" approach generally resulted in the highest metaproteome coverage; however, there are advantages to using the "online" approach when dealing with limited biomass. Both approaches resulted in a larger number of protein identifications from the synthetic metaproteome rather than from the soil metaproteome, although the soil metaproteome had a significantly larger number of predicted proteins. A large dynamic range in abundances of proteins resulting from metabolically active and inactive populations within the soil metaproteome explains this observation.
Fungi are essential to the transformation of carbon (C) and are well-known for degrading recalcitrant plant material. They are also increasingly recognized for their bioremediation potential in contaminated environments. Some fungi transform selenium (Se), an essential nutrient and toxin of growing concern, from an aqueous bioavailable phase (Se(+IV, VI)) to solid or volatile phases (Se(0, -II)). We examined the effect of C on growth and Se transformation for several Ascomycota fungi, including Alternaria alternata SRC1lrK2f, Paraphaeosphaeria sporulosa AP3s5-JAC2a and Pyrenochaeta sp. DS3sAY3a, grown in 0–60 mmol C/L glucose or acetate with 0.1 mM Se(IV) over 23 days. Higher C concentrations coincided with more Se removal, but some species preferred certain C sources. A. alternata generally reduced the most Se regardless of C source, but Pyrenochaeta sp. reduced more with glucose. A. alternata predominantly solidified Se, whereas P. sporulosa mainly volatilized Se. Glucose and acetate concentrations in cultures with and without Se trended downward throughout the experiment, suggesting that carbon consumption continues despite Se presence. As biomass did not substantially increase throughout the experiment for any organism with Se(IV) but C consumption continued, it is likely that the fungi experienced a toxicity effect with Se(IV) as observed with increased pigment production, and predominantly consumed C for cell maintenance and repair purposes. This study helps discern the effect of C on fungal Se transformations, providing greater insight into the coupling between C and Se biogeochemical cycling as well as improving Se bioremediation strategies.
The genus Paraconiothyrium has worldwide distribution with diverse host habitats and exhibits potential utilisation as biocontrol agent, bioreactor and antibiotic producer. In this review, we firstly comprehensively summarise the current taxonomic status of Paraconiothyrium species, including their category names, morphological features, habitats, and multigene phylogenetic relationships. Some Paraconiothyrium species possess vital biological functions and potential applications in medicine, agriculture, industry, and environmental protection. A total of 147 secondary metabolites have been reported so far from Paraconiothyrium, among which 95 are novel. This paper serves to provide an overview of their diverse structures with chemical classification and biological activities. To date, 27 species of Paraconiothyrium have been documented; however, only seven have been investigated for their secondary metabolites or biological functions. Our review is expected to draw more attention to this genus for providing a taxonomic reference, discovering extensive biological functions, and searching in-depth for new bioactive natural products.
Development of proteome analysis of extracellular proteins has revealed that a wide variety of proteins, including fungal allergens are present outside the cell. These secreted allergens often do not contain known secretion signal sequences. Recent research progress shows that some fungal allergens are secreted by unconventional secretion pathways, including autophagy- and extracellular-vesicle-dependent pathways. However, secretion pathways remain unknown for the majority of extracellular proteins. This review summarizes recent data on unconventional protein secretion in Saccharomyces cerevisiae and other fungi. Particularly, methods for evaluating unconventional protein secretion are proposed for fungal species, including S. cerevisiae, a popular model organism for investigating protein secretion pathways.
Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.
Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cel-lulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that syner-gistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Sta-gonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulo-sum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore , we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-PLOS ONE |
Linking community composition to ecosystem function is a challenge in complex microbial communities. We tested the hypothesis that key biological features of fungi - evolutionary history, functional guild, and abundance of functional genes - can predict the biogeochemical activity of fungal species during decay. We measured the activity of 10 different enzymes produced by 48 model fungal species on leaf litter in laboratory microcosms. Taxa included closely related species with different ecologies (i.e. species in different “functional guilds”) and species with publicly available genomes. Decomposition capabilities differed less among phylogenetic lineages of fungi than among different functional guilds. Activity of carbohydrases and acid phosphatase was significantly higher in litter colonized by saprotrophs compared to ectomycorrhizal species. By contrast, oxidoreductase activities per unit fungal biomass were statistically similar across functional guilds, with white rot fungi having highest polyphenol oxidase activity and ectomycorrhizal fungi having highest peroxidase activity. On the ecosystem level, polyphenol oxidase activity in soil correlated with the abundance of white rot fungi, while soil peroxidase activity correlated with the abundance of ectomycorrhizal fungi in soil. Copy numbers of genes coding for different enzymes explained the activity of some carbohydrases and polyphenol oxidase produced by fungi in culture, but were not significantly better predictors of activity than specific functional guild. Collectively, our data suggest that quantifying the specific functional guilds of fungi in soil, potentially through environmental sequencing approaches, allows us to predict activity of enzymes that drive soil biogeochemical cycles.
BACKGROUND: Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings.
RESULTS: It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of <= 300 aa residues, at least four cysteines, and a total cysteine content of >=5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina.
CONCLUSIONS: While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.
Fungi, as well as the rest of living organisms must deal with environmental challenges such as stressful stimuli. Fungi are excellent models to study the general mechanisms of the response to stress, because of their simple, but conserved, signal-transduction and metabolic pathways that are often equivalent to those present in other eukaryotic systems. A factor that has been demonstrated to be involved in these responses is polyamine metabolism, essentially of the three most common polyamines: putrescine, spermidine and spermine. The gathered evidences on this subject suggest that polyamines are able to control cellular signal transduction, as well as to modulate protein-protein interactions. In the present review, we will address the recent advances on the study of fungal metabolism of polyamines, ranging from mutant characterization to potential mechanism of action during different kinds of stress in selected fungal models.
The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade
cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of
selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used
to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase,
both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51
arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related
to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected
after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase,
and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent
with selective ligninolysis by C. subvermispora.
Accumulation of intracellular lipid in oleaginous yeast cells has been studied for providing an alternative supply for energy, biofuel. Numerous studies have been conducted on increasing lipid content in oleaginous yeasts. However, few explore the mechanism of the high lipid accumulation ability of oleaginous yeast strains at the proteomics level. In this study, a time-course comparative proteomics analysis was introduced to compare the non-oleaginous yeast Saccharomyces cerevisiae, with two oleaginous yeast strains, Cryptococcus albidus and Rhodosporidium toruloides at different lipid accumulation stages. Two dimensional LC-MS/MS approach has been applied for protein profiling together with isobaric tag for relative and absolute quantitation (iTRAQ) labelling method. 132 proteins were identified when three yeast strains were all at early lipid accumulation stage; 122 and 116 proteins were found respectively within cells of three strains collected at middle and late lipid accumulation stages. Significantly up-regulation or down-regulation of proteins were experienced among comparison. Essential proteins correlated to lipid synthesis and regulation were detected. Our approach provides valuable indication and better understanding for lipid accumulation mechanism from proteomics level and would further contribute to genetic engineering of oleaginous yeasts.
Fungi of the genus Pycnoporus are white-rot basidiomycetes widely studied because of their ability to synthesize high added-value compounds and enzymes of industrial interest. Here we report the sequencing, assembly and analysis of the transcriptome of Pycnoporus sanguineus BAFC 2126 grown at stationary phase, in media supplemented with copper sulfate. Using the 454 pyrosequencing platform we obtained a total of 226,336 reads (88,779,843 bases) that were filtered and de novo assembled to generate a reference transcriptome of 7,303 transcripts. Putative functions were assigned for 4,732 transcripts by searching similarities of six-frame translated sequences against a customized protein database and by the presence of conserved protein domains. Through the analysis of translated sequences we identified transcripts encoding 178 putative carbohydrate active enzymes, including representatives of 15 families with roles in lignocellulose degradation. Furthermore, we found many transcripts encoding enzymes related to lignin hydrolysis and modification, including laccases and peroxidases, as well as GMC oxidoreductases, copper radical oxidases and other enzymes involved in the generation of extracellular hydrogen peroxide and iron homeostasis. Finally, we identified the transcripts encoding all of the enzymes involved in terpenoid backbone biosynthesis pathway, various terpene synthases related to the biosynthesis of sesquiterpenoids and triterpenoids precursors, and also cytochrome P450 monooxygenases, glutathione S-transferases and epoxide hydrolases with potential functions in the biodegradation of xenobiotics and the enantioselective biosynthesis of biologically active drugs. To our knowledge this is the first report of a transcriptome of genus Pycnoporus and a resource for future molecular studies in P. sanguineus.
The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity
and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural
information is added and curated on a regular basis based on the available literature. In addition to the use of the database
by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably
a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families,
the development of subfamilies in several families and the power of CAZy for the analysis of genomes and metagenomes. This
article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the
resolution and the carbohydrate ligands in crystallographic complexes of CAZymes.
Aspergillus fumigatus Z5 has a strong ability to decompose lignocellulose biomass, and its extracellular protein secretion has been reported in earlier studies employing traditional techniques. However, a comprehensive analysis of its secretion in the presence of different carbon sources is still lacking. The goal of this work was to identify, quantify and compare the secretome of A. fumigatus Z5 in the presence of different carbon sources to understand in more details the mechanisms of lignocellulose decomposition by Aspergillus fumigatus Z5.
Cellulolytic A. fumigatus Z5 was grown in the presence of glucose (Gl), Avicel (Av) and rice straw (RS), and the activities of several lignocellulosic enzymes were determined with chromatometry method. The maximum activities of endoglucanase, exoglucanase, beta-glucosidase, laminarinase, lichenase, xylanase and pectin lyase were 12.52, 0.59, 2.30, 2.37, 1.68, 15.02 and 11.40 U.ml-1, respectively. A total of 152, 125 and 61 different proteins were identified in the presence of RS, Av and Gl, respectively, and the proteins were functionally divided into glycoside hydrolases, lipases, peptidases, peroxidases, esterases, protein translocating transporters and hypothetical proteins. A total of 49 proteins were iTRAQ-quantified in all the treatments, and the quantification results indicated that most of the cellulases, hemicellulases and glycoside hydrolases were highly upregulated when rice straw and Avicel were used as carbon sources (compared with glucose).
The proteins secreted from A. fumigatus Z5 under different carbon source conditions were identified by LC-MS/MS and quantified by iTRAQ-based quantitative proteomics. The results indicated that A. fumigatus Z5 could produce considerable cellulose-, hemicellulose-, pectin- and lignin-degrading enzymes that are valuable for the lignocellulosic bioenergy industry.
Background
Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from endogenous resources becomes mobilized to fuel maintenance and self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes.
Results
This study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential gene expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry.
Conclusions
This study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved.
Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.
A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.
Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.
Peptidases, their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database (http://merops.sanger.ac.uk) aims to fulfil the need for an integrated source of information about these. The database has hierarchical classifications
in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families,
which are in turn grouped into clans. The database has been expanded to include proteolytic enzymes other than peptidases.
Special identifiers for peptidases from a variety of model organisms have been established so that orthologues can be detected
in other species. A table of predicted active-site residue and metal ligand positions and the residue ranges of the peptidase
domains in orthologues has been added to each peptidase summary. New displays of tertiary structures, which can be rotated
or have the surfaces displayed, have been added to the structure pages. New indexes for gene names and peptidase substrates
have been made available. Among the enhancements to existing features are the inclusion of small-molecule inhibitors in the
tables of peptidase–inhibitor interactions, a table of known cleavage sites for each protein substrate, and tables showing
the substrate-binding preferences of peptidases derived from combinatorial peptide substrate libraries.
Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H(2)O(2)-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.
The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.
Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase
the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither
demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase
active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with
no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be
nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage,
and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble
cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important,
but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications
in industrial biomass utilization.
Biologically active, passive treatment systems are commonly employed for removing high concentrations of dissolved Mn(II)
from coal mine drainage (CMD). Studies of microbial communities contributing to Mn attenuation through the oxidation of Mn(II)
to sparingly soluble Mn(III/IV) oxide minerals, however, have been sparse to date. This study reveals a diverse community
of Mn(II)-oxidizing fungi and bacteria existing in several CMD treatment systems.
The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.
The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures.
The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.
Cellulose degradation by brown rot fungi, such as Postia placenta, is poorly understood relative to the phylogenetically related white rot basidiomycete, Phanerochaete chrysosporium. To elucidate the number, structure, and regulation of genes involved in lignocellulosic cell wall attack, secretome and
transcriptome analyses were performed on both wood decay fungi cultured for 5 days in media containing ball-milled aspen or
glucose as the sole carbon source. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a total of 67 and 79 proteins
were identified in the extracellular fluids of P. placenta and P. chrysosporium cultures, respectively. Viewed together with transcript profiles, P. chrysosporium employs an array of extracellular glycosyl hydrolases to simultaneously attack cellulose and hemicelluloses. In contrast,
under these same conditions, P. placenta secretes an array of hemicellulases but few potential cellulases. The two species display distinct expression patterns for
oxidoreductase-encoding genes. In P. placenta, these patterns are consistent with an extracellular Fenton system and include the upregulation of genes involved in iron
acquisition, in the synthesis of low-molecular-weight quinones, and possibly in redox cycling reactions.
Accurate and precise methods for estimating incorrect peptide and protein identifications are crucial for effective large-scale proteome analyses by tandem mass spectrometry. The target-decoy search strategy has emerged as a simple, effective tool for generating such estimations. This strategy is based on the premise that obvious, necessarily incorrect "decoy" sequences added to the search space will correspond with incorrect search results that might otherwise be deemed to be correct. With this knowledge, it is possible not only to estimate how many incorrect results are in a final data set but also to use decoy hits to guide the design of filtering criteria that sensitively partition a data set into correct and incorrect identifications.
The fungal vacuole is an extremely complex organelle that is involved in a wide variety of functions. The vacuole not only carries out degradative processes, the role most often ascribed to it, but also is the primary storage site for certain small molecules and biosynthetic precursors such as basic amino acids and polyphosphate, plays a role in osmoregulation, and is involved in the precise homeostatic regulation of cytosolic ion and basic amino acid concentration and intracellular pH. These many functions necessitate an intricate interaction between the vacuole and the rest of the cell; the vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Because of the various roles and properties of the vacuole, it has been possible to isolate mutants which are defective in various vacuolar functions including the storage and uptake of metabolites, regulation of pH, sorting and processing of vacuolar proteins, and vacuole biogenesis. These mutants show a remarkable degree of genetic overlap, suggesting that these functions are not individual, discrete properties of the vacuole but, rather, are closely interrelated.
A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn2+ to Mn3+, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations
(100 mM) of these chelators and at pH 5.0, Mn3+ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate
and malonate, but not pyrophosphate, led to H2O2 formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate,
H2O2 production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence
of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn2+ oxidation and abiotic decomposition of these organic chelators by the resulting Mn3+, which leads to formation of superoxide and its subsequent reduction to H2O2. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn3+ complexes in assays containing 1 mM Mn2+ and 100 mM oxalate or malonate, but omitting an additional H2O2 source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed
Mn2+ oxidation in providing H2O2 for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation
of lignin and xenobiotics.
In nature, cellulose, lignocellulose and lignin are major sources of plant biomass; therefore, their recycling is indispensable for the carbon cycle. Each polymer is degraded by a variety of microorganisms which produce a battery of enzymes that work synergically. In the near future, processes that use lignocellulolytic enzymes or are based on microorganisms could lead to new, environmentally friendly technologies. This study reviews recent advances in the various biological treatments that can turn these three lignicellulose biopolymers into alternative fuels. In addition, biotechnological innovations based on natural delignification and applied to pulp and paper manufacture are also outlined.
Filamentous fungi are unique organisms-rivaled only by actinomycetes and plants-in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.
The exoproteome of the fungus Fusarium graminearum grown on glucose and on hop (Humulus lupulus, L.) cell wall has been investigated. The culture medium was found to contain a higher quantity of proteins and the proteins are more diverse when the fungus is grown on cell wall. Using both 1D and 2D electrophoresis followed by mass spectrometry analysis and protein identification based on similarity searches, 84 unique proteins were identified in the cell wall-grown fungal exoproteome. Many are putatively implicated in carbohydrate metabolism, mainly in cell wall polysaccharide degradation. The predicted carbohydrate-active enzymes fell into 24 different enzymes classes, and up to eight different proteins within a same class are secreted. This indicates that fungal metabolism becomes oriented towards synthesis and secretion of a whole arsenal of enzymes able to digest almost the complete plant cell wall. Cellobiohydrolase is one of the only four proteins found both after growth on glucose and on plant cell wall and we propose that this enzyme could act as a sensor of the extracellular environment. Extensive knowledge of this very diverse F. graminearum exoproteome is an important step towards the full understanding of Fusarium/plants interactions.
The fungal vacuole is an extremely complex organelle that is involved in a wide variety of functions. The vacuole not only carries out degradative processes, the role most often ascribed to it, but also is the primary storage site for certain small molecules and biosynthetic precursors such as basic amino acids and polyphosphate, plays a role in osmoregulation, and is involved in the precise homeostatic regulation of cytosolic ion and basic amino acid concentration and intracellular pH. These many functions necessitate an intricate interaction between the vacuole and the rest of the cell; the vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Because of the various roles and properties of the vacuole, it has been possible to isolate mutants which are defective in various vacuolar functions including the storage and uptake of metabolites, regulation of pH, sorting and processing of vacuolar proteins, and vacuole biogenesis. These mutants show a remarkable degree of genetic overlap, suggesting that these functions are not individual, discrete properties of the vacuole but, rather, are closely interrelated.
Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but the software tools to analyse tandem mass spectra are lagging behind. We present a database search tool MS-GF+ that is sensitive (it identifies more peptides than most other database search tools) and universal (it works well for diverse types of spectra, different configurations of MS instruments and different experimental protocols). We benchmark MS-GF+ using diverse spectral data sets: (i) spectra of varying fragmentation methods; (ii) spectra of multiple enzyme digests; (iii) spectra of phosphorylated peptides; and (iv) spectra of peptides with unusual fragmentation propensities produced by a novel alpha-lytic protease. For all these data sets, MS-GF+ significantly increases the number of identified peptides compared with commonly used methods for peptide identifications. We emphasize that although MS-GF+ is not specifically designed for any particular experimental set-up, it improves on the performance of tools specifically designed for these applications (for example, specialized tools for phosphoproteomics).
An extensive culture-dependent and -independent study was conducted to identify microorganisms contributing to the biogeochemical cycling of manganese (Mn) in Ashumet Pond, a freshwater pond in Massachusetts currently undergoing remediation. A variety of bacteria (including Gamma-, Beta-, and Alpha-proteobacteria, Firmicutes, and Bacteroides) and Ascoymete fungi were isolated from the pond that promote Mn(II) oxidation and subsequent formation of Mn(III/IV) oxide minerals. Targeted-amplicon pyrosequencing of the bacterial and fungal communities associated with Mn oxide-encrusted samples show a highly diverse microbial community, of which the cultured phylotypes represent a minor proportion. This suggests a larger community, not identified through culturing, contributes to Mn oxide formation within the Pond.
Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mechanism is proposed, in which HHQ is considered as a monophenol or as an o-diphenol, depending on the part of the molecule that interacts with the enzyme. The kinetic parameters obtained from an analysis of the measurements of the initial steady state rate of 2-hydroxy p-benzoquinone formation were kcatapp= 229.0 ± 7.7 s(-1) and KMapp,HHQ= 0.40 ± 0.05 mM. Furthermore, the action of tyrosinase on HHQ led to the enzyme's inactivation through a suicide inactivation mechanism. This suicide inactivation process was characterized kinetically by λmaxapp (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of λmaxapp and r were (8.2 ± 0.1) × 10(-3) s(-1) and 35,740 ± 2,548, respectively. © 2014 IUBMB Life, 2014.
The autolytic process observed in fungi has been divided into three different phases A 1, A 2, A 3. Each of which is characterized by specific metabolic evants. Recently, we characterized these phases by means of 14C-d-glucose. In this communication we report on the uptake and conversion of 14C-labelled amino acids. These amino acids were rapidly taken up by the fungal mycelium and incorporated into cellular pools. During autolysis phase A 1 these amino acids were still used for protein synthesis. The autolytic process resembled a turnover process during this first phase. At this time the mycelium still exhibited high metabolic activity. The second phase, A 2 was characterized by a rapid intracellular degradation of compartmental material and accompanied by oxidation of the products formed. This was reflected by an increasing 14CO2 production. During phase A 3 products of intracellular degradation and possibly compounds from autolysing hyphae were still taken up by intact, i. e. living compartments of the hyphae and converted to heat and CO2.
Almost no radioactive material was released into the medium during the last two phases. Metabolically active compartments or parts of the hyphae were present during the whole process of autolysis of fungal batch culture, .i e. even at the end of phase A 3.
Microorganisms, although being very diverse because they comprise prokaryotic organisms such as bacteria or eukaryotic organisms such as fungi, all share an essential exodigester function. The consequence is their essential need to have a secretome adapted to their environment. The selection pressure exerted by environmental constraints led to the emergence of species with varying complexity in terms of composition of their secretomes. This review on fungal secretomes highlights the extraordinary variability among these organisms, even within the same species, and hence the absolute necessity to fully characterize all their components in the aims of understanding the fundamental mechanisms responsible for secretome plasticity and developing applications notably toward a better control of diseases caused by these pathogens.
Extracellular proteases are commonly produced among fungi including several examples of wood-degrading Basidiomycetes. It was frequently observed that high endo-l,4-β-glucanase activity was always accompanied by a high production of proteolytic enzymes when Sporotrichum pulverulentum was cultivated on cellulose. This chapter describes two acidic proteases (protease I and protease II) that have been shown to play a role in the activation of the endo-l,4-β-glucanases in culture solutions of S. pulverulentum grown on cellulose. The chapter discusses assay method and purification procedure for the proteases. The specificities of protease I and protease II are determined by analyzing the degradation products from human fibrinopeptide A. Human fibrinopeptide A is isolated under clotting conditions for highly purified human fibrinogen, contains 16 amino acid residues, and has a molecular weight of 1536.
In aging carbon-depleted cultures of Penicillium chrysogenum the fragmentation and autolysis of old mycelia were coupled with the production of high levels of extracellular protease and N-acetyl-b-D-hexosaminidase; this could be stopped by the addition of an extra dose of glucose at any incubation time tested, but was not affected by endogenous NH3. After the addition of glucose, intracellular enzyme accumulation was observed only for the N-acetyl-b-D-hexosaminidase, and the concomitant decrease in both the extracellular protease and N-acetyl-b-D-hexosaminidase activities was not caused by the action of extracellular proteases. The physiological function of the N-acetyl-b-D-hexosaminidase remains to be elucidated because the P. chrysogenum culture studied did not utilize N-acetyl-D-glucosamine as a carbon source. However, the proteases (mainly serine and, to a much smaller extent, metalloproteases) might provide surviving hyphal fragments with sufficient amino acids to maintain the cryptic growth observed in aging carbon-depleted cultures.
Many fungi degrade cellulose and hemicelluloses using extracellular hydrolytic enzymes, but fungi that degrade woody biomass
are the only ones to efficiently degrade polysaccharides encased in lignin. White-rot basidiomycetes begin by mineralizing
the lignin, using extracellular oxidative enzymes to cleave this recalcitrant biopolymer. Enzymes with likely roles include
lignin peroxidases, manganese peroxidases, versatile peroxidases and laccases. In some cases the enzyme may attack the lignin
polymer directly; in others the ligninolytic agent is likely a small molecule that one of the enzymes has oxidized to a reactive
form. So far, all white rot fungi appear to secrete manganese peroxidases, and most produce laccases, whereas the other two
enzymes are less common. After ligninolysis, white-rot fungi assimilate the remaining polysaccharides using conventional glycosylhydrolase
systems that contain both endo- and exo-acting enzymes. Brown rot basidiomycetes also degrade lignocellulose efficiently,
but their biodegradative systems are less comprehensive. These fungi generally lack ligninolytic enzymes, initiating decay
instead with reactive oxygen species generated from the reaction between Fe2+ and H2O2. The limited disruption caused by these oxidants apparently allows a limited set of endo-acting glycosylhydrolases to depolymerize
the remaining polysaccharides. Biological wood pulping is one promising application of ligninolytic fungi.
The basidiomycete Schizophyllum commune produces a variety of proteolytic enzymes. A number of these, detected in native gelatin-containing polyacrylamide gels, have their activities increased during nitrogen-limited growth of the mycelium. ScPrB, a metallo-endoprotease, appears to have the greatest nitrogen stress-induced increase in activity of all of these enzymes. Quantifying ScPrB has proven difficult because no artificial chromogenic substrate has been found. In addition, it is poorly resolved from another highly active protease, ScPrA, in native gelatin-containing gels. We have developed a method using SDS gelatin-containing polyacrylamide gels for resolving ScPrB from ScPrA and for quantifying its activity by densitometry. This method was used to assess the intramycelial location of ScPrB induction after the transfer of exponentially growing colonies to nitrogen deprivation conditions. By all analyses (proportional, normalized to fresh weight, and normalized to protein), the increase in ScPrB activity was found to occur exclusively in midsections of the growing mycelium, whereas ScPrB activity was found to be decreased or unchanged in the centers of colonies and in colony margins. This implies that proteolysis mediated by ScPrB may supply translocatable amino acids only from the region directly behind the growing hyphal apices.
In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.
Lignin is the second most abundant constituent of the cell wall of vascular plants, where it protects cellulose towards hydrolytic attack by saprophytic and pathogenic microbes. Its removal represents a key step for carbon recycling in land ecosystems, as well as a central issue for industrial utilization of plant biomass. The lignin polymer is highly recalcitrant towards chemical and biological degradation due to its molecular architecture, where different non-phenolic phenylpropanoid units form a complex three-dimensional network linked by a variety of ether and carbon-carbon bonds. Ligninolytic microbes have developed a unique strategy to handle lignin degradation based on unspecific one-electron oxidation of the benzenic rings in the different lignin substructures by extracellular haemperoxidases acting synergistically with peroxide-generating oxidases. These peroxidases poses two outstanding characteristics: (i) they have unusually high redox potential due to haem pocket architecture that enables oxidation of non-phenolic aromatic rings, and (ii) they are able to generate a protein oxidizer by electron transfer to the haem cofactor forming a catalytic tryptophanyl-free radical at the protein surface, where it can interact with the bulky lignin polymer. The structure-function information currently available is being used to build tailor-made peroxidases and other oxidoreductases as industrial biocatalysts.
Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.
Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.
The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.
Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
Two acidic proteases from the white-rot fungus Sporotrichum pulverulentum have been purified and partially characterized. The enzymes were purified in four steps, protease I 152-fold and protease II 127-fold. The purity of the enzymes was investigated by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis.
The isoelectric points of protease I and protease II were at pH 4.7 and 4.2 respectively. The molecular weights were 28000 and 26000 and the pH optima 5.0 and 5.2. Both enzymes were inhibited by heavy metal ions such as Ag+, Hg2+ and to some extent also by Cu2+. Partial inhibition was also observed with EDTA and αα′- dipyridyl.
The specificities of protease I and II were investigated using human fibrinopeptide A as substrate. Protease splits off the arginine in the C-terminal position, while protease II splits at the carboxyl side of both Phe-8 and Leu-9 in fibrinopeptide A.
A physiological effect of the two proteases has also been demonstrated. Thus, if a culture solution from S. pulverulentum grown on cellulose was treated with the individual proteases or a mixture of the enzymes a considerable increase in endo-l,4-β-glucanase activity was obtained. It may be that the endo-glucanases are produced in a zymogenic form and activated by the proteases. However, other explanations for the phenomenon are also possible.
The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.
Fungal laccases are extracellular multinuclear copper-containing oxidases that have been proposed to be involved in ligninolysis and degradation of xenobiotics. Here, we show that an electrophoretically homogenous laccase preparation from the white rot fungus Trametes versicolor oxidized Mn2+ to Mn3+ in the presence of Na-pyrophosphate, with a Km value of 186 microM and a Vmax value of 0.11 micromol/min/mg protein at the optimal pH (5.0) and a Na-pyrophosphate concentration of 100 mM. The oxidation of Mn2+ involved concomitant reduction of the laccase type 1 copper site as usual for laccase reactions, thus providing the first evidence that laccase may directly utilize Mn2+ as a substrate.
An industrial strain of Penicillium chrysogenum was subjected to carbon or nitrogen limitation in a chemostat and the response monitored in terms of the "classical" indicators of autolysis (biomass decline and ammonia release), culture degradation (as measured by image analysis) and by obtaining profiles for three classes of proteases implicated in autolysis. Under both sets of conditions (carbon or nitrogen limitation), once started, autolysis involved a succession of different protease activities. The first stages of the process of autolysis in starved chemostat cultures was associated with peaks in the activities of both serine and aspartyl proteases, coinciding with the mobilisation of endogenous energy reserves. Conversely, a peak in the activity of metalloproteases was associated with the later stages of autolysis, perhaps occurring in response to depletion of endogenous energy reserves; the activity of these enzymes led to gross culture degradation, disintegration of ordered mycelial structures and signalled the end of metabolic activity (respiration) within the culture. These findings indicate that strategies intended to control/regulate autolysis in large-scale industrial fungal cultures might profitably be focused on regulation of the activity of key classes of proteases involved in the series of events leading to culture degradation.
Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.
Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.