TAILIEUCHUNG - Báo cáo khoa học: Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii

TheX-raystructureofnativecellobiohydrolase IB(CBHIB) from the filamentous fungus Talaromyces emersonii,PDB 1Q9H,was solved A ˚ bymolecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of 60 A ˚ ·40 A ˚ ·50 A ˚ andanoverall b-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase fromthis thermophilic eukaryote. | Eur. J. Biochem. 271 4495-4506 2004 FEBS 2004 doi Three-dimensional structure of a thermostable native cellobiohydrolase CBH IB and molecular characterization of the cel7 gene from the filamentous fungus Talaromyces emersonii Alice Grassick1 I Patrick G. Murray Roisin Thompson2 Catherine M. Collins1 Lucy Byrnes3 Gabriel Birrane4 Timothy M. Higgins2 and Maria G. Tuohy1 1 Molecular Glycobiotechnology Group Department of Biochemistry department of Chemistry and 3Department of Biochemistry National University of Ireland Galway Ireland 4Beth Israel Deaconess Medical Centre Harvard Institutes of Medicine Harvard Medical School Boston MA USA The X-ray structure of native cellobiohydrolase IB CBH IB from the filamentous fungus Talaromyces emersonii PDB 1Q9H was solved to A by molecular replacement. 1Q9H is a glycoprotein that consists of a large single domain with dimensions of w 60 A X 40 A X 50 A and an overall b-sandwich structure the characteristic fold of Family 7 glycosyl hydrolases GH7 . It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H as are the catalytic residues. As a result of deletions and other changes in loop regions the binding and catalytic properties of T. emersonii 1Q9H are different. The gene cel7 encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cello-biohydrolase was virtually inactive against methylumb-eriferyl-cellobioside and chloronitrophenyl-lactoside but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emerso-nii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level. .

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