3,647 research outputs found
The investigation of type-specific features of the copper coordinating AA9 proteins and their effect on the interaction with crystalline cellulose using molecular dynamics studies
AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types
Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa
Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi, where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose and some also act on hemicelluloses, primarily other (substituted) beta-(1 -> 4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. crassa are active on both cellulose and cellulose-associated glucuronoxylan but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that NcLPMO9F preferentially cleaves xylan when acting on a cellulosebeechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, from Malbranchea cinnamomea, showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylanactive LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylanactive LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. IMPORTANCE Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain unclear. Here, we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose-glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs
Form-factors and current correlators: chiral couplings L_10(mu) and C_87(mu) at NLO in 1/N(C)
Using the resonance chiral theory Lagrangian, we perform a calculation of the
vector and axial-vector two-point functions at the next-to-leading order (NLO)
in the 1/N(C) expansion. We have analyzed these correlators within the
single-resonance approximation and have also investigated the corrections
induced by a second multiplet of vector and axial-vector resonance states.
Imposing the correct QCD short-distance constraints, one determines the
difference of the two correlators Pi(t) = Pi_VV(t)- Pi_AA(t) in terms of the
pion decay constant and resonance masses. Its low momentum expansion fixes then
the low-energy chiral couplings L_10 and C_87 at NLO, keeping full control of
their renormalization scale dependence. At mu_0=0.77 GeV, we obtain L_10(mu_0)
= (-4.4 \pm 0.9)10^{-3} and C_87^r(mu_0)=(3.1 \pm 1.1)10^{-5}
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Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.
Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies
Fungal ecological strategies reflected in gene transcription - a case study of two litter decomposers.
Microbial communities interplay with their environment through their functional traits that can be a response or an effect on the environment. Here, we explore how a functional trait-the decomposition of organic matter, can be addressed based on genetic markers and how the expression of these markers reflect ecological strategies of two fungal litter decomposer Gymnopus androsaceus and Chalara longipes. We sequenced the genomes of these two fungi, as well as their transcriptomes at different steps of Pinus sylvestris needles decomposition in microcosms. Our results highlighted that if the gene content of the two species could indicate similar potential decomposition abilities, the expression levels of specific gene families belonging to the glycoside hydrolase category reflected contrasting ecological strategies. Actually, C. longipes, the weaker decomposer in this experiment, turned out to have a high content of genes involved in cell wall polysaccharides decomposition but low expression levels, reflecting a versatile ecology compare to the more competitive G. androsaceus with high expression levels of keystone functional genes. Thus, we established that sequential expression of genes coding for different components of the decomposer machinery indicated adaptation to chemical changes in the substrate as decomposition progressed
Adoleszente IdentitÀtsbildung unter postmoderenen Lebensbedingungen: Neue Freiheit oder IdentitÀtsdiffusion?
I. Grundthesen\ud
1.) Psychoanalyse als Erfahrungswissenschaft unterliegt den historischĂÂkulturellen Bedingungen ihrer Zeit . ihre Befunde und Theoretisierungen sind Ausdruck und Symptom dieser ZeitumstĂ€nde. Eine Psychoanalyse, die sich dieser Tatsache gegenĂŒber verschlieĂt, ihre einmal gewonnenen Ergebnisse ontologisiert, wird auf ihrem kulturkritischem Auge blind und auf kurz oder lang auch auf ihrem klinischen. Die Gefahr ist die einer Naturalisierung kulturell erzeugter PhĂ€nomene. \ud
2.) Der Begriff der .IdentitĂ€t., von Erikson in die Psychoanalyse eingeĂÂbracht, ist Symptom fĂŒr eine Krise . daĂ .IdentitĂ€tsbildung. unter spĂ€tĂÂindustriellen Lebensbedingungen problematischer wird. Das IdentitĂ€tsĂÂthema bĂŒndelt in paradigmatischer Form die Folgen aktueller ModerniĂÂsierungsprozesse fĂŒr die Subjekte. \ud
3.) Der Begriff IdentitĂ€t bedarf einer grundsĂ€tzlichen Differenzierung: zu unterscheiden ist eine universell-anthropologische und eine kulturellĂÂspezifische Dimension; universell: es geht in jedem Menschenleben um das Herstellen einer Passung zwischen subjektivem Innen und dem gesellĂÂschaftlichen AuĂen, das ist die anthropologische Grundaufgabe des MenĂÂschen. Aber diese Passungsaufgabe ist in heiĂen Kulturen dramatischer. \ud
4.) Die Phase der Adoleszenz ist wie keine andere Entwicklungsphase den jeweiligen kulturellen Bedingungen ausgesetzt, geht es doch in ihr um das Verlassen des .Elternhauses. und um den Eintritt in die .GesellĂÂschaft.; folglich werden sich dort kulturelle VerĂ€nderungen am stĂ€rksten auswirken. \ud
5.) Das klassische psychoanalytische Konzept der Adoleszenz ist zu erweitern, insbesondere auf die Dimensionen des .NarziĂtischen. hin; aber auch die phasenspezifische Eingrenzung der IdentitĂ€tsproblematik auf die Adoleszenz muĂ aufgegeben werden. \ud
6.) Soziologie und Kulturwissenschaften liefern empirische Befunde zur IdentitÀtsbildung in spÀtindustriellen Gesellschaften. Die Psychoanalyse sollte diese zur Kenntnis nehmen, ihre eigenen unter hochspezifischen UmstÀnden gewonnenen Erfahrungen dazu in einen kritischen Dialog bringen
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The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.
Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level
Aggregation, Persistence and Volatility in a Macromodel
Starting from microeconomic foundations, we derive a general formula for the aggregation of outputs of heterogeneous firms (or sectors), and we solve explicitly for the fundamental intertemporal equilibrium path of the aggregate economy. The firms are subject to temporary technology shocks, but the aggregate output has radically different dynamical properties, and a special form of long memory and nonlinearity never used hitherto. We study, analytically, the implied time series properties of the new process characterizing aggregate GDP per capita. This process is more persistent than any dynamically-stable linear process (e.g. autoregressions) and yet is mean-reverting (unlike unit-root processes), and its volatility is of a greater order of magnitude than that of any of its components. This amplification of volatility means that even small shocks at the micro level can lead to large fluctuations at the macro level. The process is also characterized by long cycles which have random lengths and which are asymmetric. Increased monopoly power will tend to reduce the amplitude and increase the persistence of business cycles. Strikingly, we find that the nonlinear aggregate process has an S-shaped decay of memory, similar to the data but unlike linear time series models such as the widely-used Auto-Regressive Integrated Moving-Average (ARIMA) processes and their special cases (including fractional Integration).
Anomaly cancellation in K3 orientifolds
We study in detail the pattern of anomaly cancellation in D=6 Type IIB Z_N
orientifolds, occurring through a generalized Green-Schwarz mechanism involving
several RR antisymmetric tensors and scalars fields. The starting point is a
direct string theory computation of the inflow of anomaly arising from magnetic
interaction of D-branes, O-planes and fixed-points, which are encoded in
topological one-loop partition functions in the RR odd spin-structure. All the
RR anomalous couplings of these objects are then obtained by factorization.
They are responsible for a spontaneous breaking of U(1) factors through a Higgs
mechanism involving the corresponding hypermultiplets. Some of them are also
related by supersymmetry to gauge couplings involving the NSNS scalars sitting
in the tensor multiplets. We also comment on the possible occurrence of
tensionless strings when these couplings diverge.Comment: 39 pages, LaTex, 1 figure; minor misprints correcte
Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi
Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here, we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six T. terrestris AA9 (TtAA9) LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. TtLPMO9A and TtLPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, TtLPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. TtLPMO9A, TtLPMO9E, TtLPMO9G, and TtLPMO9T could degrade tamarind xyloglucan and, with the exception of TtLPMO9T, beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs, as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates
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