Crystal Structure of a Cellulosomal Family 3 Carbohydrate Esterase from Clostridium thermocellum Provides Insights into the Mechanism of Substrate Recognition

• Published in: Journal of molecular biology Vol. 379; no. 1; pp. 64 - 72
• Main Authors: Correia, Márcia A.S., Prates, José A.M., Brás, Joana, Fontes, Carlos M.G.A., Newman, Joseph A., Lewis, Richard J., Gilbert, Harry J., Flint, James E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 23.05.2008
• Subjects: acetyl xylan; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Binding Sites - genetics; Carboxylesterase - chemistry; Carboxylesterase - genetics; Catalysis; catalytic triad; Clostridium thermocellum; Clostridium thermocellum - enzymology; Crystallography, X-Ray; esterases; Protein Conformation; Substrate Specificity; X-ray crystallography; X-ray crystallography; PNPAc; catalytic triad; esterases; acetyl xylan; SAD
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.03.037

The microbial degradation of the plant cell wall is of increasing industrial significance, exemplified by the interest in generating biofuels from plant cell walls. The majority of plant cell-wall polysaccharides are acetylated, and removal of the acetyl groups through the action of carbohydrate esterases greatly increases the efficiency of polysaccharide saccharification. Enzymes in carbohydrate esterase family 3 (CE3) are common in plant cell wall-degrading microorganisms but there is a paucity of structural and biochemical information on these biocatalysts. Clostridium thermocellum contains a single CE3 enzyme, CtCes3, which comprises two highly homologous (97% sequence identity) catalytic modules appended to a C-terminal type I dockerin that targets the esterase into the cellulosome, a large protein complex that catalyses plant cell wall degradation. Here, we report the crystal structure and biochemical properties of the N -terminal catalytic module ( CtCes3-1) of CtCes3. The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. CtCes3-1 displays an α/β hydrolase fold that contains a central five-stranded parallel twisted β-sheet flanked by six α-helices. In addition, the enzyme contains a canonical catalytic triad in which Ser44 is the nucleophile, His208 is the acid-base and Asp205 modulates the basic nature of the histidine. The acetate moiety is accommodated in a hydrophobic pocket and the negative charge of the tetrahedral transition state is stabilized through hydrogen bonds with the backbone N of Ser44 and Gly95 and the side-chain amide of Asn124.