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The Streptomyces lividans Family 12 Endoglucanase: Construction of the Catalytic Core, Expression, and X-ray Structure at 1.75 Å Resolution

Abstract : Cellulases are the glycoside hydrolases responsible for the enzymatic breakdown of the structural plant polymer cellulose. Together with xylanases they counteract the limitless accumulation of plant biomass in nature and are of considerable fundamental and biotechnological interest. Endoglucanase CelB from Streptomyces lividans performs hydrolysis of the β-1,4-glycosidic bonds of cellulose, with net retention of anomeric configuration. The enzyme is a member of glycoside hydrolase family 12 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695−696], which had previously eluded detailed structural analysis. A truncated, but catalytically competent form of CelB, lacking the flexible linker region and cellulose-binding domain, has been constructed and overexpressed in a S. lividans expression system. The three-dimensional X-ray structure of the resulting catalytic domain, CelB2, has been solved by conventional multiple isomorphous replacement methods and refined to an R factor of 0.187 at 1.75 Å resolution. The overall fold of the enzyme shows a remarkable similarity to that of family 11 xylanases, as previously predicted by hydrophobic clustering analysis [Törrönen, A., Kubicek, C. P., and Henrissat, B. (1993) FEBS Lett. 321, 135−139]. The 23 kDa protein presents a jelly-roll topology, built up mainly by antiparallel β-sheets arranged in a sandwich-like manner. A deep substrate-binding cleft runs across the surface, as has been observed in other endoglucanase structures, and is potentially able to accommodate up to five binding subsites. The likely catalytic nucleophile and Brønsted acid/base, residues Glu 120 and Glu 203, respectively, have their carboxylate groups separated by a distance of approximately 7.0 Å and are located approximately 15 Å from one end of the cleft, implying a −3 to +2 active site
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https://hal.archives-ouvertes.fr/hal-03160736
Contributor : Gerlind Sulzenbacher Connect in order to contact the contributor
Submitted on : Friday, March 5, 2021 - 3:06:49 PM
Last modification on : Saturday, October 23, 2021 - 1:44:03 PM

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Gerlind Sulzenbacher, François Shareck, Rolf Morosoli, Claude Dupont, Gideon J Davies. The Streptomyces lividans Family 12 Endoglucanase: Construction of the Catalytic Core, Expression, and X-ray Structure at 1.75 Å Resolution. Biochemistry, American Chemical Society, 1997, 36 (51), pp.16032-16039. ⟨10.1021/bi972407v⟩. ⟨hal-03160736⟩

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