Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli.
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ABSTRACT:
SUBMITTER: Hawkins AR
PROVIDER: S-EPMC1158294 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.
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Project description:The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins.
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Project description:Telomeres and subtelomere regions have vital roles in cellular homeostasis and can facilitate niche adaptation. However, information on telomere/subtelomere structure is still limited to a small number of organisms. Prior to initiation of this project, the Neurospora crassa genome assembly contained only 3 of the 14 telomeres. The missing telomeres were identified through bioinformatic mining of raw sequence data from the genome project and from clones in new cosmid and plasmid libraries. Their chromosomal locations were assigned on the basis of paired-end read information and/or by RFLP mapping. One telomere is attached to the ribosomal repeat array. The remaining chromosome ends have atypical structures in that they lack distinct subtelomere domains or other sequence features that are associated with telomeres in other organisms. Many of the chromosome ends terminate in highly AT-rich sequences that appear to be products of repeat-induced point mutation, although most are not currently repeated sequences. Several chromosome termini in the standard Oak Ridge wild-type strain were compared to their counterparts in an exotic wild type, Mauriceville. This revealed that the sequences immediately adjacent to the telomeres are usually genome specific. Finally, despite the absence of many features typically found in the telomere regions of other organisms, the Neurospora chromosome termini still retain the dynamic nature that is characteristic of chromosome ends.
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Project description:The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed.
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Project description:L-Carnitine is a bioactive compound derived from L-lysine and S-adenosyl-L-methionine, which is closely associated with the transport of long-chain fatty acids in the intermediary metabolism of eukaryotes and sought after in the pharmaceutical, food, and feed industries. The L-carnitine biosynthesis pathway has not been observed in prokaryotes, and the use of eukaryotic microorganisms as natural L-carnitine producers lacks economic viability due to complex cultivation and low titers. While biotransformation processes based on petrochemical achiral precursors have been described for bacterial hosts, fermentative de novo synthesis has not been established although it holds the potential for a sustainable and economical one-pot process using renewable feedstocks. This study describes the metabolic engineering of Escherichia coli for L-carnitine production. L-carnitine biosynthesis enzymes from the fungus Neurospora crassa that were functionally active in E. coli were identified and applied individually or in cascades to assemble and optimize a four-step L-carnitine biosynthesis pathway in this host. Pathway performance was monitored by a transcription factor-based L-carnitine biosensor. The engineered E. coli strain produced L-carnitine from supplemented L-N ε-trimethyllysine in a whole cell biotransformation, resulting in 15.9 μM carnitine found in the supernatant. Notably, this strain also produced 1.7 μM L-carnitine de novo from glycerol and ammonium as carbon and nitrogen sources through endogenous N ε-trimethyllysine. This work provides a proof of concept for the de novo L-carnitine production in E. coli, which does not depend on petrochemical synthesis of achiral precursors, but makes use of renewable feedstocks instead. To the best of our knowledge, this is the first description of L-carnitine de novo synthesis using an engineered bacterium.
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Project description:Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.
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Project description:To determine the genes directly and indirectly under the control of the Grainy-head homolog (GHH) transcription factor in Neurospora crassa
2012-03-01 | GSE35017 | GEO
Project description:Cryptochromes were identified in plants and animals where they function as either photoreceptors or circadian clock components. In the filamentous fungus Neurospora, the biological function of cryptochrome has not yet been explored. Here, we demonstrate that Neurospora crassa cryptochrome (Nc cry) is a DASH-type of cryptochrome, capable of binding FAD and MTHF, whose transcript and protein levels are both strongly induced by blue light in a wc-1 dependent manner. Although the Nc cry transcript is circadian-regulated and antiphasic to frq, knockout strains of Nc cry appears to have a normal clock phenotype. Whole genome microarray and RT-QPCR analysis confirm that Nc cry is not involved in the signal transduction of either early or late light responses and seems to have no transcriptional regulatory activity under our laboratory conditions. Our study concludes that the only cryptochrome in Neurospora crassa is dispensable for the well-characterized blue light sensing cascade and is not part of the circadian clock system. Keywords: light response
2010-03-31 | GSE14909 | GEO
Project description:Cryptochromes were identified in plants and animals where they function as either photoreceptors or circadian clock components. In the filamentous fungus Neurospora, the biological function of cryptochrome has not yet been explored. Here, we demonstrate that Neurospora crassa cryptochrome (Nc cry) is a DASH-type of cryptochrome, capable of binding FAD and MTHF, whose transcript and protein levels are both strongly induced by blue light in a wc-1 dependent manner. Although the Nc cry transcript is circadian-regulated and antiphasic to frq, knockout strains of Nc cry appears to have a normal clock phenotype. Whole genome microarray and RT-QPCR analysis confirm that Nc cry is not involved in the signal transduction of either early or late light responses and seems to have no transcriptional regulatory activity under our laboratory conditions. Our study concludes that the only cryptochrome in Neurospora crassa is dispensable for the well-characterized blue light sensing cascade and is not part of the circadian clock system. Keywords: light response Two-color microarray. Alexa Fluor 555 was consistently used to label cDNA synthesized from reference RNA, which is a mixture containing equal amounts of RNA samples harvested from different circadian time points and light treatment durations. The same batch of pooled RNA was used as a reference for each array experiment. Alexa Fluor 647 was used exclusively to label cDNA representing sample RNA.
2010-06-05 | E-GEOD-14909 | biostudies-arrayexpress