Structural Insights into the -Xylosidase from Trichoderma reesei Obtained by Synchrotron Small-Angle X-ray Scattering and Circular Dichroism Spectroscopy - Analytical Methods & Tools for Lyophilization

Structural Insights into the -Xylosidase from Trichoderma reesei Obtained by Synchrotron Small-Angle X-ray Scattering and Circular Dichroism Spectroscopy
Received May 4, 2005
Revised Manuscript Received August 13, 2005
Web Release Date: November 5, 2005
Adriana L. Rojas, Hannes Fischer, Elena V. Eneiskaya, Anna A. Kulminskaya, Konstantin A. Shabalin, Kirill N. Neustroev, Aldo F. Craievich, Alexander M. Golubev, and Igor Polikarpov*
Biochemistry
ACS Publications
Copyright ? 2005 American Chemical Society
Instituto de F?sica de S?o Carlos, Universidade de S?o Paulo, Av. Trabalhador S?o-carlense 400, CEP 13560-970, S?o Carlos, SP, Brazil, Petersburg Nuclear Physics Institute, Gatchina, St. Petersburg, 188300 R?ssia, and Instituto de F?sica, Universidade de S?o Paulo, S?o Paulo, SP, Brazil
Abstract:
The enzyme -xylosidase from Trichoderma reesei, a member of glycosil hydrolase family 3 (GH3), is a glycoside hydrolase which acts at the glycosidic linkages of 1,4--xylooligosaccharides and that also exhibits -L-arabinofuranosidase activity on 4-nitrophenyl -L-arabinofuranoside. In this work, we show that the enzyme forms monomers in solution and derive the low-resolution molecular envelope of the -xylosidase from small-angle X-ray scattering (SAXS) data using the ab initio simulated annealing algorithm. The radius of gyration and the maximum dimension of the -xylosidase are 30.3 ? 0.2 and 90 ? 5 ?, respectively. In contrast to the fold of the only two structurally characterized members of GH3, the barley -D-glucan exohydrolase and -hexosaminidase from Vibrio cholerae, which have respectively two or one distinct domains, the shape of the -xylosidase indicates the presence of three distinct structural modules. Domain recognition algorithms were used to show that the C-terminal part of the amino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs demonstrate that this additional domain adopts a predominantly conformation.
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