Crystallization and preliminary X-ray crystallographic analysis of a novel α-L-arabinofuranosidase (CtGH43) from Clostridium thermocellum ATCC 27405.

The truncated carbohydrate-active enzyme belonging to family 43 glycoside hydrolase from Clostridium thermocellum (CtGH43) is an α-L-arabinofuranosidase that in combination with endoxylanase leads to complete breakdown of L-arabinosyl-substituted xylans. The recombinant enzyme CtGH43 from C. thermocellum was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. The recombinant CtGH43 has a molecular mass of 35.86 kDa. Preliminary structural characterization was carried out on CtGH43 crystallized from different conditions, which gave either cube-shaped or brick-shaped crystals. These diffracted to a resolution of 1.65 Å for the cubic form and 1.1 Å for the monoclinic form. Molecular replacement was used to solve the CtGH43 structure.

[1]  Pedro M. Coutinho,et al.  The carbohydrate-active enzymes database (CAZy) in 2013 , 2013, Nucleic Acids Res..

[2]  M. N. Gupta,et al.  A Novel α-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf) from Clostridium thermocellum , 2013, PloS one.

[3]  K. Diederichs,et al.  Better models by discarding data? , 2013, Acta crystallographica. Section D, Biological crystallography.

[4]  D. Goyal,et al.  Bioethanol Production Involving Recombinant C. thermocellum Hydrolytic Hemicellulase and Fermentative Microbes , 2012, Applied Biochemistry and Biotechnology.

[5]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[6]  Owen Johnson,et al.  iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM , 2011, Acta crystallographica. Section D, Biological crystallography.

[7]  Philip R. Evans,et al.  An introduction to data reduction: space-group determination, scaling and intensity statistics , 2011, Acta crystallographica. Section D, Biological crystallography.

[8]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[9]  Makoto Yoshida,et al.  Isolation and Characterization of CcAbf62A, a GH62 α-L-Arabinofuranosidase, from the Basidiomycete Coprinopsis cinerea , 2011, Bioscience, biotechnology, and biochemistry.

[10]  H. Brumer,et al.  The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases* , 2011, The Journal of Biological Chemistry.

[11]  J. François,et al.  Characterization of the family GH54 α-l-arabinofuranosidases in Penicillium funiculosum, including a novel protein bearing a cellulose-binding domain , 2010, Applied Microbiology and Biotechnology.

[12]  Graeme Winter,et al.  xia2: an expert system for macromolecular crystallography data reduction , 2010 .

[13]  G. Volckaert,et al.  Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family. , 2009, The Biochemical journal.

[14]  Fei Long,et al.  BALBES: a molecular-replacement pipeline , 2007, Acta crystallographica. Section D, Biological crystallography.

[15]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[16]  H. R. Sørensen,et al.  A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan , 2006, Applied Microbiology and Biotechnology.

[17]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[18]  Lei Chang,et al.  Biochemical and kinetic characterization of GH43 β-d-xylosidase/α-l-arabinofuranosidase and GH30 α-l-arabinofuranosidase/β-d-xylosidase from rumen metagenome , 2011, Journal of Industrial Microbiology & Biotechnology.