The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

Coordinated Regulation of Membrane Homeostasis and Drug Accumulation by Novel Kinase STK-17 in Response to Antifungal Azole Treatment.

The emergence of antifungal resistance, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, makes fungal infections difficult to treat in clinics and agriculture. When exposed to azoles, fungi can make adaptive responses to alleviate azole toxicity and produce azole tolerance. However, except for azole efflux pumps and ergosterol biosynthesis genes, the role of most azole responsive genes in azole resistance is unknown. In this study, STK-17, whose transcription is upregulated by azoles, was characterized as a novel kinase that is required for azole resistance. Deletion or dysfunction of STK-17 led to azole hypersensitivity in Neurospora crassa and to other ergosterol biosynthesis inhibitors such as amorolfine, terbinafine, and amphotericin B, but not fatty acid and ceramide biosynthesis inhibitors. STK-17 was also required for oxidative stress resistance, but this was not connected to azole resistance. RNA-seq results showed that stk-17 deletion affected the basal expression and the response to ketoconazole of some membrane protein genes, indicating functional association of STK-17 with the membrane. Notably, deletion of stk-17 affected the normal response to azoles of erg genes, including the azole target-encoding gene erg11, and erg2, erg6, and erg24, and led to abnormal accumulation of sterols in the presence of azoles. HPLC-MS/MS analysis revealed increased intracellular azole accumulation in the stk-17 mutant, possibly due to enhanced azole influx and reduced azole efflux that was independent of the major efflux pump CDR4. Importantly, STK-17 was widely distributed and functionally conserved among fungi, thus providing a potential antifungal target. IMPORTANCE Antifungal resistance is increasing worldwide, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, making control of fungal infections more challenging. A lot of effort has been expended in elucidating the mechanism of azole resistance and revealing potential antifungal targets. In this study, by analyzing azole-responsive genes in Neurospora crassa, we discovered STK-17, a novel kinase, that is required for azole resistance in several types of fungi. It has a role in regulating membrane homeostasis, responses to azole by ergosterol biosynthesis genes and azole accumulation, thus, deepening our understanding on the mechanism of azole stress response. Additionally, STK-17 is conserved among fungi and plays important roles in fungal development and stress resistance. Kinase inhibitors are broadly used for treating diseases, and our study pinpoints a potential drug target for antifungal development.

Fast and Specific Peroxygenase Reactions Catalyzed by Fungal Mono-Copper Enzymes.

The copper-dependent lytic polysaccharide monooxygenases (LPMOs) are receiving attention because of their role in the degradation of recalcitrant biomass and their intriguing catalytic properties. The fundamentals of LPMO catalysis remain somewhat enigmatic as the LPMO reaction is affected by a multitude of LPMO- and co-substrate-mediated (side) reactions that result in a complex reaction network. We have performed kinetic studies with two LPMOs that are active on soluble substrates, NcAA9C and LsAA9A, using various reductants typically employed for LPMO activation. Studies with NcAA9C under "monooxygenase" conditions showed that the impact of the reductant on catalytic activity is correlated with the hydrogen peroxide-generating ability of the LPMO-reductant combination, supporting the idea that a peroxygenase reaction is taking place. Indeed, the apparent monooxygenase reaction could be inhibited by a competing H2O2-consuming enzyme. Interestingly, these fungal AA9-type LPMOs were found to have higher oxidase activity than bacterial AA10-type LPMOs. Kinetic analysis of the peroxygenase activity of NcAA9C on cellopentaose revealed a fast stoichiometric conversion of high amounts of H2O2 to oxidized carbohydrate products. A kcat value of 124 ± 27 s-1 at 4 °C is 20 times higher than a previously described kcat for peroxygenase activity on an insoluble substrate (at 25 °C) and some 4 orders of magnitude higher than typical "monooxygenase" rates. Similar studies with LsAA9A revealed differences between the two enzymes but confirmed fast and specific peroxygenase activity. These results show that the catalytic site arrangement of LPMOs provides a unique scaffold for highly efficient copper redox catalysis.

An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme.

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.

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) ß-(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 cellulose-beechwood 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 xylan-active 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 xylan-active 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.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

Biochemical and biophysical characterization of the RVB-1/RVB-2 protein complex, the RuvBL/RVB homologues in Neurospora crassa.

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Circadian Clock Control of Translation Initiation Factor eIF2α Activity Requires eIF2γ-Dependent Recruitment of Rhythmic PPP-1 Phosphatase in Neurospora crassa.

The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of N. crassa PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of N. crassa eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that N. crassa eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events.IMPORTANCE Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus Neurospora crassa regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, N. crassa eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

The Predicted Mannosyltransferase GT69-2 Antagonizes RFW-1 To Regulate Cell Fusion in Neurospora crassa.

Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony establishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, glycosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function mutations in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus suppressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial germination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2 All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation.IMPORTANCE Cell wall remodeling is a dynamic process that balances cell wall integrity versus cell wall dissolution. In filamentous fungi, cell wall dissolution is required for somatic cell fusion and conidial separation during asexual sporulation. In the filamentous fungus Neurospora crassa, allorecognition checkpoints regulate the cell fusion process between genetically different cells. Our study revealed two linked loci with transspecies polymorphisms and under coevolution, rfw-1 and gt69-2, which form a coordinated system to regulate cell wall remodeling during somatic cell fusion, conidial separation, and asexual spore germination. RFW-1 acts as a negative regulator of these three processes, while GT69-2 functions antagonistically to RFW-1. Our findings provide new insight into the mechanisms involved in regulation of fungal cell wall remodeling during growth and development.

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2 O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2 O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2 O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2 O2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H2 O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2 O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2 O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.

ATP utilization by a DEAD-box protein during refolding of a misfolded group I intron ribozyme.

DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting nonproductive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ∼90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ∼10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with protein chaperones.

Biochemical characterization of the dicing activity of Dicer-like 2 in the model filamentous fungus Neurospora crassa.

Dicing of double-stranded RNA (dsRNA) into small RNA is an essential process to trigger transcriptional and post-transcriptional gene silencing. Using cell-free extracts of the model filamentous fungus Neurospora crassa, we successfully detected the dicing activity of one of two N. crassa Dicers NcDCL2. The predominant 23-nucleotide (nt) cleavage product was always detected from 30-nt to 130-nt dsRNA substrates, and additional products of approximately 18 to 28 nt were occasionally produced. The enzymatic properties of NcDCL2 are different from those of insect and plant small interfering RNA (siRNA)-producing Dicers, Drosophila melanogaster Dicer-2 and Arabidopsis thaliana DCL3 and DCL4 (AtDCL3 and AtDCL4). Whereas AtDCL3 and AtDCL4 preferentially cleave short and long dsRNAs, respectively, NcDCL2 cleaved both short and long dsRNAs. These results suggest that N. crassa has a single siRNA-producing Dicer NcDCL2, which is a prototype of plant siRNA-producing Dicers with distinct functions in diverse RNA silencing pathways. The dicing assay reported here is convenient to detect and biochemically characterize the dicing activities of both plant and fungal Dicers, and is likely applicable to other organisms.

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa.

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS-) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures.

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam rapidly induces radiation damage of photosensitive cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (> 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

PRD-2 directly regulates casein kinase I and counteracts nonsense-mediated decay in the Neurospora circadian clock.

Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock's positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.

Kinetic solvent viscosity effects reveal a protein isomerization in the reductive half-reaction of Neurospora crassa class II nitronate monooxygenase.

Nitronate monooxygenase from Neurospora crassa (NcNMO) is an FMN-dependent enzyme that oxidizes nitronates. The enzyme belongs to Group H of flavin monooxygenases. Previous biochemical and mechanistic studies on the enzyme showed that NcNMO oxidizes both anionic nitronates and neutral nitroalkanes, a feature distinguishing NcNMO from bacterial and other fungal NMOs which are active exclusively on nitronates. Recently, NMOs have been shown to oxidize propionate 3-nitronate (P3N), a toxic nitro acid present in legumes, fungi, and leaf beetles; P3N is an irreversible inhibitor of succinate dehydrogenase causing anywhere from neurological disorders to death in livestock and humans. In this study, we report the first kinetic investigation of NcNMO with P3N and its conjugated acid 3-nitropropionic acid (3NPA), and a mechanistic investigation with 3NPA using kinetic solvent viscosity effects. The kcat value with P3N (300 s-1) was 7-times larger than with 3NPA and the kcat/KP3N value (1,700,000 M-1s-1) was ~500-times larger than the kcat/K3NPA value, consistent with P3N being a faster and better substrate than 3NPA. The normalized kcat and kcat/K3NPA values showed inverse hyperbolic dependences on the relative solvent viscosity, consistent with an internal isomerization of the enzyme-substrate complex. A similar inverse hyperbolic pattern of the normalized kred value for the rate constant of flavin reduction determined anaerobically in a stopped-flow spectrophotometer suggested that the internal enzyme isomerization occurs before the flavin reduction step in the reductive half-reaction.

Functional dissection of gamma-aminobutyric acid metabolism in Neurospora crassa.

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.

Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple ß-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.