Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-Arabinofuranosidase.
The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (Ka of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (Ka of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant Ka of 230 M(-1) and binding capacity (N0) of 11 µmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India. arungoyl@iitg.ernet.in.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D014908:Triticum / Q000378:metabolism; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D014990:Xylans; C085118:arabinoxylan; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase