Crystal Structures of A. acidocaldarius Endoglucanase Cel9A in Complex with Cello-Oligosaccharides: Strong − 1 and − 2 Subsites Mimic Cellobiohydrolase Activity |
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Authors: | Kelvin Eckert Leila Lo Leggio |
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Affiliation: | 1 Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France 2 Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark |
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Abstract: | Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with β-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 Å resolution and two complexes at 2.66 and 2.1 Å resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (α/α)6-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites − 1 and − 2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides. |
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Keywords: | AaCel9A, Alicyclobacillus acidocaldarius Cel9A CcCel9M, Clostridium cellulolyticum Cel9M CcCel9G, Clostridium cellulolyticum Cel9G CtCel9A, Clostridium thermocellum Cel9A TfCel9A, Thermobifida fusca Cel9A GH9, glycoside hydrolase family 9 MPD, (4S)-2-methyl-2,4-pentanediol NtEG, Nasutitermes takasagoensis endoglucanase pNP, p-nitrophenyl Ig, immunoglobulin PDB, Protein Data Bank CMC, carboxymethylcellulose Glc, Glucose |
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