Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
- MeSH
- celulosa chemie MeSH
- fungální proteiny chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hmotnostní spektrometrie MeSH
- katalytická doména MeSH
- katalýza MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- kyslík chemie MeSH
- lignin chemie MeSH
- měď chemie MeSH
- Neurospora crassa enzymologie MeSH
- oxidační stres MeSH
- oxidoreduktasy chemie MeSH
- oxygenasy se smíšenou funkcí chemie MeSH
- polysacharidy chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin.
- MeSH
- chromatin metabolismus MeSH
- epigenetická represe * MeSH
- histonlysin-N-methyltransferasa metabolismus MeSH
- histony metabolismus MeSH
- lysin metabolismus MeSH
- metylace MeSH
- Neurospora crassa enzymologie genetika metabolismus MeSH
- posttranslační úpravy proteinů * MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.
- MeSH
- aminohydrolasy chemie genetika metabolismus MeSH
- Arthrodermataceae enzymologie MeSH
- Aspergillus niger enzymologie MeSH
- druhová specificita MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- fylogeneze MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny mandlové metabolismus MeSH
- Nectria enzymologie MeSH
- Neurospora crassa enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE OF WORK: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- Aspergillus niger enzymologie genetika MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fungální proteiny genetika metabolismus MeSH
- genom fungální MeSH
- Gibberella enzymologie genetika MeSH
- klonování DNA MeSH
- Neurospora crassa enzymologie genetika MeSH
- nitrily metabolismus MeSH
- organické látky metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
At a concentration of 0.5 to 3 mmol/l, ATP stimulates the activity of mitochondrial DNA polymerase of Neurospora crassa under the optimum reaction conditions; at higher concentrations, an inhibitory effect is observed. 4-Chloromercuribenzoate (1 mmol/L), a thiol inhibitor, decreases the enzyme activity two-fold, while N-ethylmalcimide (2 mmol/L) has no effect. Ethidium bromide (up to 10 mumol/L) and heparin (up to 0.4 micrograms/mL) reduce the activity by 60%. ddTTP does not affect the DNA polymerase reaction. The best in vitro template is the activated calf-thymus DNA.
- MeSH
- adenosintrifosfát farmakologie MeSH
- aktivace enzymů účinky léků MeSH
- dideoxynukleotidy MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- ethidium farmakologie MeSH
- heparin farmakologie MeSH
- inhibitory syntézy nukleových kyselin * MeSH
- mitochondrie enzymologie MeSH
- Neurospora crassa účinky léků enzymologie MeSH
- sulfhydrylová reagencia farmakologie MeSH
- thiminnukleotidy farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
