PMID(sorted descending)
genetic and molecular characterization of a cryptochrome from the filamentous fungus neurospora plants and animals, cryptochromes function as either photoreceptors or circadian clock components. we have examined the cryptochrome from the filamentous fungus neurospora crassa and demonstrate that neurospora cry encodes a dash-type cryptochrome that appears capable of binding flavin adenine dinucleotide (fad) and methenyltetrahydrofolate (mthf). the cry transcript and cry protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulat ...201020305004
visualization of f-actin localization and dynamics with live cell markers in neurospora crassa.filamentous actin (f-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. we visualized f-actin organization and dynamics in living neurospora crassa cells via confocal microscopy of growing hyphae expressing gfp fusions with homologues of the actin-binding proteins fimbrin (fim) and tropomyosin (tpm-1), a subunit of the arp2/3 complex (arp-3) and a recently developed ...201020302965
live-cell imaging of microtubule dynamics in hyphae of neurospora crassa.due to the large number of microtubules in the wild-type strain, total internal reflection fluorescence (tirf) microscopy was used to study cortical microtubule dynamics in leading hyphae of neurospora crassa expressing beta-tubulin-gfp. detection of plus-end dynamics of individual microtubule was much improved with this approach compared to the other commonly used methods such as confocal and widefield fluorescence microscopy. in order to address the roles of motor proteins in microtubule dynam ...201020238275
biochemical methods used to study the gene expression and protein complexes in the filamentous fungus neurospora crassa.biochemical approaches are powerful tools for investigating mechanisms of biological processes. here, we describe several biochemical approaches that have been successfully in our laboratory to study the filamentous fungus neurospora crassa. these approaches include protein extraction and western blot analysis, protein purification using epitope-tagged fusion protein, protein immunoprecipitation (ip) and chromatin immunoprecipitation (chip) assays. these methods can also be modified for use in o ...201020238270
gene silencing for functional analysis: assessing rnai as a tool for manipulation of gene expression.the availability of a large number of gene-disrupted mutants (either from natural mutants' collections or from knockout projects) is a great advantage for functional analysis studies. however, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose ...201020238262
high-throughput construction of gene deletion cassettes for generation of neurospora crassa knockout strains.the availability of complete genome sequences for a number of biologically important fungi has become an important resource for fungal research communities. however, the functions of many open reading frames (orfs) identified through annotation of whole genome sequences have yet to be determined. the disruption of orfs is a practical method for loss-of-function gene analyses in fungi that are amenable to transformation. unfortunately, the construction of knockout cassettes using traditional dige ...201020238259
nuclear dynamics, mitosis, and the cytoskeleton during the early stages of colony initiation in neurospora crassa.neurospora crassa macroconidia form germ tubes that are involved in colony establishment and conidial anastomosis tubes (cats) that fuse to form interconnected networks of conidial germlings. nuclear and cytoskeletal behaviors were analyzed in macroconidia, germ tubes, and cats in strains that expressed fluorescently labeled proteins. heterokaryons formed by cat fusion provided a rapid method for the imaging of multiple labeled fusion proteins and minimized the potential risk of overexpression a ...201020207852
septum formation is regulated by the rho4-specific exchange factors bud3 and rgf3 and by the landmark protein bud4 in neurospora crassa.rho gtpases have multiple, yet poorly defined functions during cytokinesis. by screening a neurospora crassa knock-out collection for rho guanine nucleotide exchange factor (gef) mutants that phenocopy rho-4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of bud3p and rgf3 of budding and fission yeast respectively. the function of these proteins as rho4-specific gefs was determined by in vitro assays. in viv ...201020199606
a single mutation in the first transmembrane domain of yeast cox2 enables its allotopic expression.during the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. to further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast cox2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. nuclear-recoded saccharomyces cerevisiae cox2 fused at the amino terminus to various alternati ...201020194738
the role of paaac1 encoding a mitochondrial adp/atp carrier in the biosynthesis of extracellular glycolipids, mannosylerythritol lipids, in the basidiomycetous yeast pseudozyma antarctica.pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (mel), which show not only excellent surface-active properties but also versatile biochemical actions. a gene homologous with a mitochondrial adp/atp carrier was dominantly expressed in p. antarctica under mel-producing conditions on the basis of previous gene expression analysis. the gene encoding the mitochondrial adp/atp carrier of p. antarctica (paaac1) contained a putative open r ...201020146402
f-actin dynamics in neurospora crassa.this study demonstrates the utility of lifeact for the investigation of actin dynamics in neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. lifeact is a 17-amino-acid peptide derived from the nonessential saccharomyces cerevisiae actin-binding protein abp140p. fused to green fluorescent protein (gfp) or red fluorescent protein (tagrfp), lifeact allowed live-cell imaging of actin patches, cable ...201020139238
the dmm complex prevents spreading of dna methylation from transposons to nearby genes in neurospora crassa.transposable elements are common in genomes and must be controlled. many organisms use dna methylation to silence such selfish dna, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. we identified a jmjc domain protein in neurospora, dna methylation modulator-1 (dmm-1), that prevents aberrant spreading of dna and histone h3k9 methylation from inactivated transposons into nearby genes. mutation of a conserved residue within the jmjc fe(ii)-binding site ab ...201020139222
systematic overrepresentation of dna termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear dna molecules.analysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. we surmised that the problem might lie in the dna shearing conditions used to create clone libraries for genome sequencing.201020122283
[phylogenetic analysis and detection of selective pressure on the cvnh domains].cyanovirin-n (cv-n) is a novel protein with broad-spectrum antiviral activity. its homologs constitute a protein family known as cvnh (cyanovirin-n homology), which possess the evolutionarily conserved anti-hiv (human immunodeficiency virus) domain. in this study, more details about the patchy organism distribution of cvnh domain were explored by reconstructing gene trees. duplicated cvnh sequences were also identified in a wide range of species including aspergillus niger, neosartorya fischeri ...201020085891
adenylyl cyclase regulates heavy metal sensitivity, bikaverin production and plant tissue colonization in fusarium proliferatum.a homologue of the adenylyl cyclase (ac) gene of neurospora crassa, named fpacy1 was cloned from the genomic library of fusarium proliferatum item 2287 by screening the library with a dna fragment amplified by using pcr primers designed from conserved sequences of the catalytic domain of ac genes from other fungi. the deduced fpacy1 protein had 53-77% identity with the ac proteins of other fungi. deltafpacy1 mutants obtained by targeted gene disruption showed retarded vegetative growth, increase ...201020082366
circadian rhythms in neurospora crassa: dynamics of the clock component frequency visualized using a fluorescent reporter.the frequency (frq) gene of neurospora crassa has long been considered essential to the function of this organism's circadian rhythm. increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. to address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins ...201020051268
parp is involved in replicative aging in neurospora crassa.modification of proteins by the addition of poly(adp-ribose) is carried out by poly(adp-ribose) polymerases (parps). parps have been implicated in a wide range of biological processes in eukaryotes, but no universal function has been established. a study of the aspergillus nidulans parp ortholog (prpa) revealed that the protein is essential and involved in dna repair, reminiscent of findings using mammalian systems. we found that a neurospora parp orthologue (npo) is dispensable for cell surviva ...201020045739
identification and biochemical characterization of molybdenum cofactor-binding proteins from arabidopsis thaliana.the molybdenum cofactor (moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. among eukaryotes, very little is known about the processes taking place subsequent to moco biosynthesis, i.e. moco transfer, allocation, and insertion into molybdenum enzymes. in the model plant arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind moco with k(d) ...200920040598
characterization of ctr family genes and the elucidation of their role in the life cycle of neurospora crassa.transcriptional analysis using qrt-pcr of 62 metal ion transporters during conidial germination of neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the ctr family. herein we characterised the ctr family genes (tcu-1, tcu-2 and tcu-3) and deciphered their role in various developmental phases of the n. crassa life cycle. cross complementation assays in copper uptake mutant of saccharomyces cerevisiae revealed that tcu-1, tcu-2 a ...201020034585
regulation by blue light of the fluffy gene encoding a major regulator of conidiation in neurospora crassa.the development of asexual spores, that is, the process of conidiation, in the fungus neurospora crassa is increased by light. the fluffy (fl) gene, encoding a major regulator of conidiation, is activated by light. we describe here a detailed characterization of the regulation by blue light of fl in vegetative hyphae. this induction requires the white collar complex (wcc) while the fld protein acts as a dark repressor of fl transcription. we show that the wcc directly regulates fl transcription ...201020026679
systems analysis of plant cell wall degradation by the model filamentous fungus neurospora crassa.the filamentous fungus neurospora crassa is a model laboratory organism, but in nature is commonly found growing on dead plant material, particularly grasses. using functional genomics resources available for n. crassa, which include a near-full genome deletion strain set and whole genome microarrays, we undertook a system-wide analysis of plant cell wall and cellulose degradation. we identified approximately 770 genes that showed expression differences when n. crassa was cultured on ground misc ...200920018766
the ndr kinase dbf-2 is involved in regulation of mitosis, conidial development, and glycogen metabolism in neurospora crassa.neurospora crassa dbf-2 encodes an ndr (nuclear dbf2-related) protein kinase, homologous to lats1, a core component of the hippo pathway. this pathway plays important roles in restraining cell proliferation and promoting apoptosis in differentiating cells. here, we demonstrate that dbf-2 is involved in three fundamental processes in a filamentous fungus: cell cycle regulation, glycogen biosynthesis, and conidiation. dbf-2 is predominantly localized to the nucleus, and most (approximately 60%) db ...201019966031
the s helix mediates signal transmission as a hamp domain coiled-coil extension in the narx nitrate sensor from escherichia coli the nitrate-responsive, homodimeric narx sensor, two cytoplasmic membrane alpha-helices delimit the periplasmic ligand-binding domain. the hamp domain, a four-helix parallel coiled-coil built from two alpha-helices (hd1 and hd2), immediately follows the second transmembrane helix. previous computational studies identified a likely coiled-coil-forming alpha-helix, the signaling helix (s helix), in a range of signaling proteins, including eucaryal receptor guanylyl cyclases, but its function re ...201019966007
the mitochondrial porin, vdac, has retained the ability to be assembled in the bacterial outer membrane.beta-barrel proteins are present in the outer membranes (oms) of gram-negative bacteria, mitochondria, and chloroplasts. their assembly requires a machinery of which the central component, called omp85 (bama) in bacteria and tob55 (sam50) in mitochondria, is evolutionarily conserved. an open question is whether the signals in beta-barrel om proteins required for assembly via this multicomponent machinery are also conserved. to address this question, we have expressed in escherichia coli the mito ...201019959601
ubiquitin ligase components cullin4 and ddb1 are essential for dna methylation in neurospora crassa.dna methylation and h3k9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. in the filamentous fungus neurospora crassa, it has been demonstrated that h3k9 trimethylation catalyzed by histone methyltransferase dim-5 is essential for dna methylation. trimethylated h3k9 is recognized by hp1, which then recruits the dna methyltransferase dim-2 to methylate the dna. here, we show that in neurospora, ubiquitin ligase components cullin4 and ddb1 are essent ...201019948733
reversible phosphorylation subserves robust circadian rhythms by creating a switch in inactivating the positive element.reversible phosphorylation of proteins is ubiquitous in circadian systems, but the role it plays in generating rhythmicity is not completely understood. a common mechanism for most circadian rhythms involves a negative feedback loop between the positive and negative elements. here, we built a minimal model for the neurospora crassa circadian clock based on the core negative feedback loop and the protein frequency (frq)-dependent phosphorylation of the white collar complex (wcc). the model can re ...200919948115
aoso protein accumulates at the septal pore in response to various stresses in the filamentous fungus aspergillus oryzae.filamentous ascomycetes form hyphal networks that are compartmentalized by septa which have a perforated pore allowing the passage of cytoplasm and organelles between adjacent hyphal compartments. thus, the septal pore may play an important role in the organized growth of multicellular organisms. upon hyphal injury, the septal pore is plugged by a wound-healing organelle, known as the woronin body, to prevent excessive cytoplasmic leakage. however, the movement of proteins towards the septal por ...201019945422
the neurospora peptide:n-glycanase ortholog png1 is essential for cell polarity despite its lack of enzymatic activity.secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. the cytoplasmic peptide:n-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 s proteasome. in contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:n-glycanase activity remains unclear. here we show that the png1 ortholog from the filamentous ...201019940117
a complex photoreceptor system mediates the regulation by light of the conidiation genes con-10 and con-6 in neurospora crassa.genes con-10 and con-6 in neurospora crassa are activated during conidiation or after illumination of vegetative mycelia. light activation requires the white-collar complex (wcc), a transcription factor complex composed of the photoreceptor wc-1 and its partner wc-2. we have characterized the photoactivation of con-10 and con-6, and we have identified 300bp required for photoactivation in the con-10 promoter. a complex stimulus-response relationship for con-10 and con-6 photoactivation suggested ...201019932184
characterization of gpit-1 and gpit-2, two auxiliary components of the neurospora crassa gpi transamidase complex.the glycosylphosphatidylinositol (gpi) transamidase contains five known subunits and functions in the lumen of the er to produce gpi-anchored proteins. the transamidase cleaves proteins containing a gpi anchor attachment signal at their c terminus and generates an amide bond between the newly generated carboxyl terminus of the protein and a gpi anchor. we have identified and characterized gpit-1 and gpit-2, two of the transamidase subunits from neurospora crassa. gpit-1 and gpit-2 are homologs o ...200919927742
protein roles in group i intron rna folding: the tyrosyl-trna synthetase cyt-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics.the neurospora crassa cyt-18 protein is a mitochondrial tyrosyl-trna synthetase that also promotes self-splicing of group i intron rnas by stabilizing the functional structure in the conserved core. cyt-18 binds the core along the same surface as a common peripheral element, p5abc, suggesting that cyt-18 can replace p5abc functionally. in addition to stabilizing structure generally, p5abc stabilizes the native conformation of the tetrahymena group i intron relative to a globally similar misfolde ...201019913030
neurospora sees the light: light signaling components in a model system.light is a key environmental signal for most life on earth. over 5% of neurospora crassa genes are expressed in response to light stimulation in a temporally regulated cascade that includes several transcription factors. fungal genomes, including neurospora's, may encode several different proteins capable of binding chromophores with the ability to harvest light energy as well as proteins that can interact with primary photoreceptors or further propogate the light signal. the best understood pho ...200919907715
snornas from the filamentous fungus neurospora crassa: structural, functional and evolutionary insights.snornas represent an excellent model for studying the structural and functional evolution of small non-coding rnas involved in the post-transcriptional modification machinery for rrnas and snrnas in eukaryotic cells. identification of snornas from neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snorna genes in the fungus kingdom.200919895704
oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion.cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. to facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. however, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self ...200919884508
camp signaling pathway controls glycogen metabolism in neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation.the camp-pka signaling pathway plays an important role in many biological processes including glycogen metabolism. in this work we investigated its role in the neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the pka pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of pka therefore is a strain with constitutively ac ...201019883780
separation of mitochondrial membranes of neurospora crassa: i. localization of l-kynurenine-3-hydroxylase. 197119866780
self-signalling and self-fusion in filamentous fungi.the formation of interconnected hyphal networks is central to the organisation and functioning of the filamentous fungal colony. it is brought about by the fusion of specialised hyphae during colony initiation and mature colony development. these hyphae are normally genetically identical, and hence this process is termed hyphal self-fusion. the conidial anastomosis tube (cat) functions in forming networks of conidial germlings during colony initiation. this hyphal type in neurospora crassa is be ...200919864177
functional stratification of the spitzenkörper of neurospora (ncu04189) is a protein required for the synthesis of beta-1,3-glucan in neurospora crassa. as chitin, beta-1,3-glucan is a morphogenetically relevant component of the fungal cell wall. previously, we showed that chitin synthases are delivered to the growing hyphal tip of n. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the spitzenkörper (spk). tagged with the green fluorescent protein (gfp), gs-1 accumulated in the hyphal apex forming a ...200919843220
cell elongation and branching are regulated by differential phosphorylation states of the nuclear dbf2-related kinase cot1 in neurospora crassa.dysfunction of the neurospora crassa nuclear dbf2-related kinase cot1 leads to cessation of tip extension and massive induction of new sites of growth. to determine the role phosphorylation plays in cot1 function, we mutated cot1 residues corresponding to positions of highly conserved nuclear dbf2-related phosphorylation sites. analyses of the point-mutation cot-1 strains (mimicking non- and constitutively phosphorylated states) indicate the involvement of cot1 phosphorylation in the regulation ...200919818014
structure and distribution of organelles and cellular location of calcium transporters in neurospora crassa.we wanted to examine the cellular locations of four neurospora crassa proteins that transport calcium. however, the structure and distribution of organelles in live hyphae of n. crassa have not been comprehensively described. therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. we observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated memb ...200919801418
circadian rhythms in neurospora crassa: downstream effectors.the circadian rhythm in neurospora crassa is exhibited as alternating areas of conidiating and non-conidiating mycelia growth. a significant role in this circadian rhythm is played by the frq (frequency) and wc (white-collar) genes, comprising the "fwc" oscillator. strains lacking the fwc can be restored to rhythmicity, which has been attributed to a second oscillator, called the flo (frq-less oscillator). this study reports additional conditions that allow this rhythmicity to occur. rhythmicity ...201019800017
two ndr kinase-mob complexes function as distinct modules during septum formation and tip extension in neurospora crassa.ndr kinases are important for growth and differentiation and require interaction with mob proteins for activity and function. we characterized the ndr kinases and mob activators in neurospora crassa and identified two ndr kinases (cot1 and dbf2) and four mob proteins (mob1, mob2a, mob2b and mob3/phocein) that form two functional ndr-mob protein complexes. the mob1-dbf2 complex is not only essential for septum formation in vegetative cells and during conidiation, but also functions during sexual ...200919788544
ptk2 contributes to osmoadaptation in the filamentous fungus neurospora crassa.hyphal tip-growing organisms often rely upon an internal hydrostatic pressure (turgor) to drive localized expansion of the cell. regulation of the turgor in response to osmotic shock is mediated primarily by an osmotic map kinase cascade which activates osmolyte synthesis and ion uptake to effect turgor recovery. we characterized a neurospora crassa homolog (ptk2) of ser/thr kinase regulators of ion transport in yeast to determine its role in turgor regulation in a filamentous fungi. the ptk2 mu ...200919772928
distinct changes in soybean xylem sap proteome in response to pathogenic and symbiotic microbe interactions.plant systemic signaling characterized by the long distance transport of molecules across plant organs involves the xylem and phloem conduits. root-microbe interactions generate systemic signals that are transported to aerial organs via the xylem sap. we analyzed the xylem sap proteome of soybean seedlings in response to pathogenic and symbiotic interactions to identify systemic signaling proteins and other differentially expressed proteins.200919772575
nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in neurospora crassa.a nucleoside diphosphate kinase-1-disrupted (ndk-1(rip-1)) mutant was observed to be defective in aerial hyphal and conidial development. in this study, two types of hyphae, fine and thick, were observed in wild-type (wt) strains. however, only fine-type hyphae were observed in the ndk-1(rip-1) mutants. the ndk-1(rip-1) mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (cat)-3. furthermore the ndk-1(rip-1) mutants could form thick hyphae by o ...200919765588
post-translational modifications in circadian rhythms.the pace has quickened in circadian biology research. in particular, an abundance of results focused on post-translational modifications (ptms) is sharpening our view of circadian molecular clockworks. ptms affect nearly all aspects of clock biology; in some cases they are essential for clock function and in others, they provide layers of regulatory fine-tuning. our goal is to review recent advances in clock ptms, help make sense of emerging themes, and spotlight intriguing (and perhaps controve ...200919740663
negative roles of a novel nitrogen metabolite repression-related gene, tar1, in laccase production and nitrate utilization by the basidiomycete cryptococcus neoformans.the multicopper oxidase laccase is widespread in fungi and has great industrial importance. one puzzle regarding laccase production in the basidiomycetous yeast cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37 degrees c). in this paper, we report identification of a nitrogen metabolite repression-related gene, tar1, which is responsible for laccase repression. disruption of tar1 results in a significant increase in the level of lac1 mrna at 37 degrees c. the putative ...200919734333
purification and characterization of a low molecular weight endo-xylanase from mushroom termitomyces clypeatus.a low molecular weight endo-xylanase (ec was purified from an edible mushroom termitomyces clypeatus grown in submerged medium with oat spelt xylan. xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kda. the enzyme was found to be most active at 50 degrees c and ph 5.0, being most stab ...201019728166
[application of bioinformatics analysis of signal peptide in the identification of neurospora crassa phya gene].to identify the function of the gene encoding neurospora crassa eaa33149.1 protein which has 46.85% similarity with aspergillus niger pha gene.200919726332
illuminating solution responses of a lov domain protein with photocoupled small-angle x-ray scattering.the pas-lov domain is a signal-transducing component found in a large variety of proteins that is responsible for sensing different stimuli such as light, oxygen, and voltage. the lov protein vvd regulates blue light responses in the filamentous fungi neurospora crassa. using photocoupled, time-resolved small-angle x-ray scattering, we extract the solution protein structure in both dark-adapted and light-activated states. two distinct dark-adapted conformations are detected in the wild-type prot ...200919712683
synthesis and antifungal activity of beta-trifluoroalkyl aminovinyl ketone derivatives.ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on several phytopathogenic fungi, an oomycete and plants were assessed. the various compounds were fungitoxic at the 10-100 microm range, with (z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en-1-one exhibiting the highest inhibitory effect on most of the test pathogens. alternaria alternata and neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. we pr ...200919711891
optimization of coprogen production in neurospora crassa.coprogen production of neurospora crassa was dependent on glucose, aspartate and iron contents as well as on initial ph of the culture media. surplus iron and acidic ph hindered the production of coprogen as well as the transcription of the sid1 gene (ncu07117) encoding putative l-ornithine-n5-monooxygenase, the first enzyme in the coprogen biosynthetic pathway. high glucose (40 g/l) and aspartate (21 g/l) concentrations were beneficial for coprogen synthesis, but neither glucose nor aspartate a ...200919700391
transcriptional profiling and functional analysis of heterokaryon incompatibility in neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death.heterokaryon incompatibility (hi) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. nonself recognition and hi occurs during hyphal fusion between strains that differ at het loci. if two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (pcd). incompatible heterokaryons s ...200919696111
hydrothermal processing and enzymatic hydrolysis of sorghum bagasse for fermentable carbohydrates production.untreated and hydrothermally treated sorghum bagasse (sb) was hydrolyzed to simple sugars by the synergistic action of cellulases and hemicellulases produced by the fungi fusarium oxysporum and neurospora crassa. synergism between the two lignocellulolytic systems was maximized with the application of higher fraction of n. crassa enzymes. hydrothermolysis of sb was studied at a wide range of treatment times and temperatures. at intense pretreatment conditions (210 degrees c for 20 min; logr(0)=4 ...200919692234
observing meiosis in filamentous fungi: sordaria and neurospora.the filamentous fungi neurospora crassa and sordaria macrospora are materials of choice for recombination studies because each of the dna strands involved in meiosis can be visually analyzed using spore-color mutants. well-advanced molecular genetic methodologies have been developed for each of these fungi, and several mutants defective in recombination and/or pairing are available. moreover, the complete genome sequence of n. crassa has made it possible to clone virtually any gene involved in t ...200919685321
cytoplasmic bulk flow propels nuclei in mature hyphae of neurospora crassa.we used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of neurospora crassa whose nuclei expressed histone h1-tagged green fluorescent protein (gfp). in addition to the h1-gfp wild-type (wt) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1, and a ro-1 kin-1 double mutant). we also treated the wt strain with benomyl and cytochalasin a to disrupt microtubules and actin microfi ...200919684281
kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.a series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for hansenula mrakii and neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. a linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pk(is)versus the ...200919683782
expression of ribonuclease a and ribonuclease n1 in the filamentous fungus neurospora this study, we investigated the ability of the fungus neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine rnase a and the endogenous rnase n(1). a set of expression vectors was constructed, each consisting of an rnase a open reading frame under the control of a specific promoter and each with a specific terminator. n. crassa transformants were analyzed at the transcriptional and protein levels. irrespective of the promoter used, all transformants showed an rnas ...201019662399
structure and applications of a rhamnolipid surfactant produced in soybean oil waste.soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by pseudomonas aeruginosa lbi strain. the chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. the chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. the monorhamnolipid rhac10c1) and the dirhamnolipid rha2c10c10 were the main components of the mixture that showed predominanc ...201019649781
deletion and overexpression of the aspergillus nidulans gata factor areb reveals unexpected pleiotropy.the aspergillus nidulans transcription factor area is a key regulator of nitrogen metabolic gene expression. area contains a c-terminal gata zinc finger dna-binding domain and activates expression of genes necessary for nitrogen acquisition. previous studies identified areb as a potential negative regulator of nitrogen catabolism showing similarity with penicillium chrysogenum nreb and neurospora crassa asd4. the areb gene encodes multiple products containing an n-terminal gata zinc finger and a ...200919628561
transcription of the neurospora crassa 70-kda class heat shock protein genes is modulated in response to extracellular ph changes.heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. these proteins are linked to a myriad of physiological functions. in this study, we show that the n. crassa hsp70-1 (ncu09602.3) and hsp70-2 (ncu08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30 degrees c. no significant accumulation of these transcripts was detected at alkaline ph values. both genes accumulated t ...201019618296
nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis.nitronate monooxygenase (nmo), formerly referred to as 2-nitropropane dioxygenase, is an fmn-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. over the past 5 years, a resurgence of interest on the enzymology of nmo has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. this revi ...201019577534
cell-free protein synthesis energized by slowly-metabolized maltodextrin.cell-free protein synthesis (cfps) is a rapid and high throughput technology for obtaining proteins from their genes. the primary energy source atp is regenerated from the secondary energy source through substrate phosphorylation in cfps.200919558718
enzymatic synthesis of bis-5-alkylresorcinols by resorcinol-producing type iii polyketide enzyme systems responsible for the biosynthesis of structurally and biosynthetically intriguing bis-5-alkylresorcinols produced by plants have been identified. herein, we show that bacterial, fungal and plant alkylresorcinol-producing type iii polyketide synthases (pkss), such as arsb in the gram-negative bacterium azotobacter vinelandii, oras in the fungus neurospora crassa and aras2 in the rice plant oryza sativa, can synthesize bis-5-alkylresorcinol from alkanedioic acid n-acetylcysteamine ...200919557027
trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi neurospora crassa and candida albicans.cell wall proteins from purified candida albicans and neurospora crassa cell walls were released using trifluoromethanesulfonic acid (tfms) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. the cell wall proteins were then characterized by sds-page and identified by proteomic analysis. the analyses for c. albicans identified 15 cell wall proteins and six secreted proteins. for n. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. mos ...200919555771
transcriptional analysis of the response of neurospora crassa to phytosphingosine reveals links to mitochondrial function.treatment of neurospora crassa cells with phytosphingosine (phs) induces programmed cell death (pcd) by an unknown mechanism. to determine the relationship between phs treatment and pcd, we determined changes in global gene expression levels in n. crassa during a time-course of phs treatment. most genes having differential expression levels compared to untreated samples showed an increase in relative expression level upon phs exposure. however, genes encoding mitochondrial proteins were highly e ...200919520721
evolutionary roles of upstream open reading frames in mediating gene regulation in fungi.upstream open reading frames (uorfs) are frequently present in the 5'-leader regions of fungal mrnas. they can affect translation by controlling the ability of ribosomes that scan from the mrna 5' end to reach the downstream genic reading frame. the translation of uorfs can also affect mrna stability. for several genes, including saccharomyces cerevisiae gcn4, s. cerevisiae cpa1, and neurospora crassa arg-2, regulation by uorfs controls expression in response to specific physiological signals. t ...200919514854
the fate of gene duplicates in the genomes of fungal pathogens.understanding how molecular changes underlie phenotypic variation within and between species is one of the main goals of evolutionary biology and comparative genetics. the recent proliferation of sequenced fungal genomes offers a unique opportunity to start elucidating the extreme phenotypic diversity in the kingdom fungi.1-4 we attempted to investigate the contribution of gene families to the evolutionary forces shaping the diversity of pathogenic lifestyles among the fungi.5 we studied a famil ...200819513258
investigation of regulatory factors required for alternative oxidase production in neurospora crassa.alternative oxidase (aox) has been found in a large number of filamentous fungi and yeasts with the notable exceptions of saccharomyces cerevisiae and schizosaccharomyces pombe. in virtually all of these fungi, aox is induced by stresses on the cell that compromise the efficiency of the standard mitochondrial electron transport chain. as aox is encoded in the nucleus and the signals that induce its expression originate in mitochondria, induction of the enzyme provides a classic example of retrog ...200919493307
properties of the kinesin-3 nckin3 motor domain and implications for neck function.members of the kinesin-3 family are microtubule motors involved in the transport of membranous cargo. nckin3 from the fungus neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. to determine how one of the heads is inactivated, we investigated truncated monomeric constructs. none of the constructs generated processive single-molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. the kineti ...200919490122
a phylogenetically conserved dna damage response resets the circadian clock.the mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. we have previously shown that dna damage can reset the circadian clock in a time-of-day-dependent manner in the filamentous fungus neurospora crassa through the modulation of negati ...200919465696
relationship between phylogenetic distribution and genomic features in neurospora the post-genome era, insufficient functional annotation of predicted genes greatly restricts the potential of mining genome data. we demonstrate that an evolutionary approach, which is independent of functional annotation, has great potential as a tool for genome analysis. we chose the genome of a model filamentous fungus neurospora crassa as an example. phylogenetic distribution of each predicted protein coding gene (pcg) in the n. crassa genome was used to classify genes into six mutually e ...200919461939
qirna is a new type of small interfering rna induced by dna damage.rna interference pathways use small rnas to mediate gene silencing in eukaryotes. in addition to small interfering rnas (sirnas) and micrornas, several types of endogenously produced small rnas have important roles in gene regulation, germ cell maintenance and transposon silencing. the production of some of these rnas requires the synthesis of aberrant rnas (arnas) or pre-sirnas, which are specifically recognized by rna-dependent rna polymerases to make double-stranded rna. the mechanism for arn ...200919444217
structure of the channels in the outer mitochondrial membrane: electron microscopic studies of the periodic arrays induced by phospholipase a(2) treatment of the neurospora membrane.the channel proteins in outer membranes of neurospora crassa mitochondria spontaneously organize into periodic arrays when the membranes are dialyzed in the presence of soluble phospholipase a(2). electron microscopic images have been recorded at different electron doses from channel arrays in a variety of negative stains, as well as in vitreous ice. fourier or correlation averages are formed from image fields containing a few hundred unit cells. these averages can be subsequently classified by ...198619431637
effects of divalent metal ions and chelators on the structure of outer mitochondrial membranes from neurospora crassa. 198219431484
activity of the circadian transcription factor white collar complex is modulated by phosphorylation of sp-motifs.posttranslational modifications, particularly phosphorylation, regulate activity, stability and localization of proteins in circadian clocks, thereby contributing to a stable oscillation with a period of approximately 24h. the white collar complex (wcc) is the central transcription factor of the circadian clock of neurospora crassa. its activity is regulated in a circadian manner by rhythmic phosphorylation, mediated by the clock protein frequency (frq). here we present purification of tap-tagge ...200919427309
the mitochondrial proteome database: mitop2.defining the mitochondrial proteome is a prerequisite for fully understanding the organelles function as well as mechanisms underlying mitochondrial pathology. the core functions of mitochondria include oxidative phosphorylation, amino acid metabolism, fatty acid oxidation, and ion homeostasis. in addition to these well-known functions, many crucial properties in cell signaling, cell differentiation and cell death are only now being elucidated, and with them the proteins involved. with the wealt ...200919426859
minimum criteria for dna damage-induced phase advances in circadian rhythms.robust oscillatory behaviors are common features of circadian and cell cycle rhythms. these cyclic processes, however, behave distinctively in terms of their periods and phases in response to external influences such as light, temperature, nutrients, etc. nevertheless, several links have been found between these two oscillators. cell division cycles gated by the circadian clock have been observed since the late 1950s. on the other hand, ionizing radiation (ir) treatments cause cells to undergo a ...200919424508
neurospora crassa fmf-1 encodes the homologue of the schizosaccharomyces pombe ste11p regulator of sexual development.the neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. we localized fmf-1 to the lg il genome segment between the centromere-proximal breakpoint of the chromosome segment duplication dp(il)39311 and the centromere. by mapping crossovers with respect to rflp markers in this region we further localized ...200919417542
molecular view of the interaction between iota-carrageenan and a phospholipid film and its role in enzyme immobilization.proteins incorporated into phospholipid langmuir-blodgett (lb) films are a good model system for biomembranes and enzyme immobilization studies. the specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. in a model system, instead, lb film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. in this ...200919415915
unwinding by local strand separation is critical for the function of dead-box proteins as rna chaperones.the dead-box proteins cyt-19 in neurospora crassa and mss116p in saccharomyces cerevisiae are broadly acting rna chaperones that function in mitochondria to stimulate group i and group ii intron splicing and to activate mrna translation. previous studies showed that the s. cerevisiae cytosolic/nuclear dead-box protein ded1p could stimulate group ii intron splicing in vitro. here, we show that ded1p complements mitochondrial translation and group i and group ii intron splicing defects in mss116de ...200919393667
alterations in growth and branching of neurospora crassa caused by sub-inhibitory concentrations of antifungal agents.six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in neurospora crassa. among the antifungals herein used, the azole agent ketoconazole at 0.5 microg/ml inhibited radial growth more than fluconazole at 5.0 microg/ml while amphotericin b at 0.05 microg/ml was more effective than nystatin at 0.05 microg/ml. morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine ...201419391525
chitosan permeabilizes the plasma membrane and kills cells of neurospora crassa in an energy dependent manner.chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, neurospora crassa. rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabili ...200919389478
growth at high ph and sodium and potassium tolerance in media above the cytoplasmic ph depend on ena atpases in ustilago maydis.potassium and na(+) effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. in fungal cells, these effluxes are mediated by cation/h(+) antiporters and ena atpases. we have cloned and studied the functions of the two ena atpases of ustilago maydis, u. maydis ena1 (umena1) and umena2. umena1 is a typical k(+) or na(+) efflux atpase whose function is indispensable for growth at ph 9.0 and for even modest na(+) or k(+) tolerances above ph 8.0. umena1 locates to ...200919363061
the basidiomycetous mushroom lentinula edodes white collar-2 homolog phrb, a partner of putative blue-light photoreceptor phra, binds to a specific site in the promoter region of the l. edodes tyrosinase gene.we isolated a cdna homolog of neurospora crassa wc-2 from the basidiomycetous mushroom lentinula edodes and termed it phrb cdna. the deduced phrb (313 amino acid residues) contained a pas domain and a zinc-finger motif. random binding-site selection analysis of the phrb produced in escherichia coli revealed that it bound to a 7-bp sequence with the consensus sequence 5'gata/ttg/t/ac3'. electrophoretic mobility-shift assay showed that it also bound to the consensus sequence 5'gatattc3' in the pro ...200919344689
a calcium binding protein from cell wall of neurospora crassa.isolated cell wall preparations of n. crassa bind significant levels of ca, mg and other divalent cations. enzymatic treatment of the cell wall with beta-(1,3)-glucanase, but not with chitinase, resulted in solubilization of only the calcium-binding protein fraction. a calcium-binding protein (cabp) was purified by metal-chelate affinity chromatography and reversed phase hplc. cabp has an mr of around 6 kda on sds-page and mass spectrometry showed that it has a molecular mass of 5744 da. one mol ...200919322841
re-characterisation of saccharomyces cerevisiae ach1p: fungal coa-transferases are involved in acetic acid detoxification.saccharomyces cerevisiae and neurospora crassa mutants defective in the so-called acetyl-coa hydrolases ach1p and acu-8, respectively, display a severe growth defect on acetate, which is most strongly pronounced under acidic conditions. acetyl-coa hydrolysis is an energy wasting process and therefore denoted as a biochemical conundrum. acetyl-coa hydrolases show high sequence identity to the coa-transferase coat from aspergillus nidulans. therefore, we extensively re-characterised the yeast enzy ...200919298859
vaccine production in neurospora crassa.we have chosen to use the filamentous fungus neurospora crassa to produce subunit vaccines. here we describe the production and purification of influenza hemagglutinin and neuraminidase antigens in n. crassa. the n. crassa system used by neugenesis offers many advantages over other systems for production of recombinant protein. in contrast to mammalian cell culture, n. crassa can be grown in a rapid and economic manner, generating large amounts of recombinant protein in simple, defined medium. v ...200919285427
the polarisome component spa-2 localizes at the apex of neurospora crassa and partially colocalizes with the spitzenkö fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. polarity allows the directional traffic of vesicles to the spitzenkörper (spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. one of these complexes is the polarisome, which in saccharomyces cerevisiae contains spa2p, pea2p, bud6p/aip3p and bni1p. to investigate the localization and role of ...200919281855
carotenoids and carotenogenic genes in podospora anserina: engineering of the carotenoid composition extends the life span of the mycelium.carotenoids have been identified in the fungus podospora anserina and a parallel pathway to neurosporene and beta-carotene was established. three genes for the beta-carotene branch have been cloned and their function elucidated. they correspond to the al-1, al-2 and al-3 genes from neurospora crassa. they were individually and in combinations over-expressed in p. anserina in order to modify the carotenoid composition qualitatively and quantitatively. in the resulting transformants, carotenoid sy ...200919277665
a novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi sordaria macrospora and neurospora crassa.during fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete sordaria macrospora. real time pcr analysis showed that the genes from the orthologous cluster in neurospora crassa are also upregulated during development. the cluster encodes putative polyketide biosynthesis enzymes, including a reducing ...200919277664
genome-wide analysis of light-inducible responses reveals hierarchical light signalling in neurospora.white collar-1 (wc-1) and white collar-2 (wc-2) are essential for light-mediated responses in neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and putative photoreceptors remain poorly characterized. unsupervised hierarchical clustering of genome-wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. evidence is shown for photoreception in the absence of the d ...200919262566
transient responses during hyperosmotic shock in the filamentous fungus neurospora crassa.fungal cells maintain an internal hydrostatic pressure (turgor) of about 400-500 kpa. in the filamentous fungus neurospora crassa, the initial cellular responses to hyperosmotic treatment are loss of turgor, a decrease in relative hyphal volume per unit length (within 1 min) and cell growth arrest; all recover over a period of 10-60 min due to increased net ion uptake and glycerol production. the electrical responses to hyperosmotic treatment are a transient depolarization of the potential (with ...200919246761
quelling targets the rdna locus and functions in rdna copy number control.rna silencing occurs in a broad range of organisms. although its ancestral function is probably related to the genome defense mechanism against repetitive selfish elements, it has been found that rna silencing regulates different cellular processes such as gene expression and chromosomal segregation. in neurospora crassa, a rna silencing mechanism, called quelling, acts to repress the expression of transgenes and transposons, but until now no other cellular functions have been shown to be regula ...200919243581
effects of mitochondrial complex iii disruption in the respiratory chain of neurospora mitochondria from most organisms, including neurospora crassa, dimeric complex iii was found associated with complex i. additional association of complex iv with this core structure leads to the formation of a respirasome. it was recently described for bacteria and mammals that complex iii is needed for the assembly/stability of complex i. to elucidate the role of complex iii in the organization of the respiratory chain of n. crassa, we analysed strains devoid of either the rieske iron-sulphu ...200919239619
the bem46-like protein appears to be essential for hyphal development upon ascospore germination in neurospora crassa and is targeted to the endoplasmic reticulum.the bud emergence (bem)46 proteins are evolutionarily conserved members of the alpha/beta-hydrolase super family, but their exact role remains unknown. to better understand the cellular role of bem46 and its homologs, we used the model organism neurospora crassa in conjunction with bem46 rnai, over-expression vectors, and repeat induced point mutation analyzes. we clearly demonstrated that bem46 is required for cell type-specific hyphal growth, which indicates a role for bem46 in maintaining pol ...200919238386
catabolite repression directly affects transcription of the qa-y gene of neurospora crassa.the quinic acid (qa) gene cluster of neurospora crassa is subject to two levels of gene control: a primary system which responds to the presence of quinic acid via the qa-1s repressor protein, and a secondary system which represses transcription of qa genes in the presence of a preferred carbon source, e.g. glucose. rna blot analysis revealed that transcription of the qa-y gene, which encodes a quinic acid permease, was strongly repressed in the presence of glucose even in the absence of the qa- ...200919236936
transcriptional changes in the nuc-2a mutant strain of neurospora crassa cultivated under conditions of phosphate shortage.the molecular mechanism that controls the response to phosphate shortage in neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the preg-pgov complex. this allows the translocation of the transcriptional factor nuc-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. the nuc-2a mutant strain of n. crassa carries a loss-of-function mu ...200919230635
chromosome segment duplications in neurospora crassa: barren crosses beget fertile science.studies on neurospora chromosome segment duplications (dps) performed since the publication of perkins's comprehensive review in 1997 form the focus of this article. we present a brief summary of perkins's seminal work on chromosome rearrangements, specifically, the identification of insertional and quasiterminal translocations that can segregate dp progeny when crossed with normal sequence strains (i.e., t x n). we describe the genome defense process called meiotic silencing by unpaired dna tha ...200919204993
inflated kinetic isotope effects in the branched mechanism of neurospora crassa 2-nitropropane dioxygenase.catalytic turnover of neurospora crassa 2-nitropropane dioxygenase with nitroethane as substrate occurs through both nonoxidative and oxidative pathways. the ph dependence of the kinetic isotope effects with [1,1-(2)h(2)]nitroethane as substrate was measured in the current study by monitoring the formation of the nitronate product in the nonoxidative pathway. the kinetic isotope effect on the second-order rate constant for nitronate formation, k(cat)/k(m), decreased from an upper limiting value ...200919199786
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