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Background Fungal beta-glucosidases (BGs) from glucoside hydrolase family 3 (GH3) are industrially important enzymes, which convert cellooligosaccharides into glucose; the end product of the cellulolytic process. They are highly active against the β-1,4 glycosidic bond in soluble substrates but typically reported to be inactive against insoluble cellulose. Results We studied the activity of four fungal GH3 BGs on cellulose and found significant activity. At low temperatures (10 ℃), we derived the approximate kinetic parameters kcat = 0.3 ± 0.1 s−1 and KM = 80 ± 30 g/l for a BG from Aspergillus fumigatus (AfBG) acting on Avicel. Interestingly, this maximal turnover is higher than reported values for typical cellobiohydrolases (CBH) at this temperature and comparable to those of endoglucanases (EG). The specificity constant of AfGB on Avicel was only moderately lowered compared to values for EGs and CBHs. Conclusions Overall these observations suggest a significant promiscuous side activity of the investigated GH3 BGs on insoluble cellulose. This challenges the traditional definition of a BG and supports suggestions that functional classes of cellulolytic enzymes may represent a continuum of overlapping modes of action.

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDUl's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG! expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant logl-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the GdulD phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Amyloid-β peptide (Aβ) seems to have a central role in the neuropathology of Alzheimer's disease (AD) 1 . Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes 2 , 3 . Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Aβ 42 ) 4 , 5 , 6 , 7 , 8 , which is the predominant form found in the amyloid plaques of Alzheimer's disease 9 , 10 . The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner 11 , 12 . In the present study, transgenic animals were immunized with Aβ 42 , either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-β deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of β-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-β may be effective in preventing and treating Alzheimer's disease.

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Biochemical analysis of protein function provides important mechanistic information. However, a full understanding of a protein's function additionally requires a three dimensional structure of the protein. This work investigates the structure-function relationship of four different proteins. Two α/β hydrolase enzymes are (MsJHE) is involved in inactivation of an insect hormone and the other (Bna7p) is a yeast enzyme involved in the synthesis of tryptophan metabolites. Another enzyme in the tryptophan metabolism pathway, Bna3p, is investigated. It is a PLP dependent enzyme that catalyzes the transamination of kynurenine and various α-keto acids. Finally, a second insect protein, the odorant binding protein AgamOBP, is examined. The MsJHE structure reveals an unusual binding pocket consisting of a long, hydrophobic tunnel, which ends just beyond the catalytic serine. The structure clearly explains the enzyme's preference for substrate esters generated from long, unbranched hydrophobic acids and small alcohols. The structure also reveals the presence of a conserved aromatic residue that interacts with the sulfur atom of the inhibitor OTFP. Mutational analysis demonstrates that this aromatic residue partly explains the increased potency gained by adding a sulfur atom to an alkyl trifluoromethyl ketone inhibitor. The crystal structure of Bna7p had previously been determined, but its function was unknown. The structure has features consistent with the protein using N-formyl kynurenine, which is made from an aromatic amide and a small acid, as a substrate. This enzyme was determined through further biochemical and genetic evidence to be the S. cerevisiae N-formyl kynurenine formamidase. Bna3p had originally been thought to have this function. However, examination of the crystal structure of the enzyme, long with an understanding of the PLP-dependent aminotransferase catalytic mechanism made this designation unlikely. This work provides structural and biochemical evidence that Bna3p is the yeast kynurenine aminotransferase. Finally, the crystal structure of the odorant binding protein from Anopheles gambiae reveals an unusual, long hydrophobic tunnel (much like that found in MsJHE). A large number of potentially acid labile bonds in the C-terminus of the protein, along with an observation of pH dependent changes in binding and structure, suggest a mechanism of ligand release.

Amyloid-b peptide (Ab) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes,. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Ab sub(42)), which is the predominant form found in the amyloid plaques of Alzheimer's disease,. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner,. In the present study, transgenic animals were immunized with Ab sub(42), either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-b deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of b-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-b may be effective in preventing and treating Alzheimer's disease.