title: Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition.
abstract: 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 alpha/beta hydrolase fold that contains a central five-stranded parallel twisted beta-sheet flanked by six alpha-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.
authors.affiliation: CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
authors.forename: Márcia A S
authors.initials: MA
authors.lastname: Correia
authors.forename: José A M
authors.initials: JA
authors.lastname: Prates
authors.forename: Joana
authors.initials: J
authors.lastname: Brás
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Newman
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: James E
authors.initials: JE
authors.lastname: Flint
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D018360:Crystallography, X-Ray; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D043182:Carboxylesterase