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The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a ␣/␤ hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 Å resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a ␤-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co 2؉ . Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" Many plant cell wall polysaccharides, including xylan, mannan, and pectin are present in acetylated forms. Acetylation not only modifies the physicochemical properties of polysaccharides, notably increasing the solubility for matrix applications, but also means they are less readily attacked by phytopathogen-derived cell wall-degrading endoglycosidases. To overcome the steric problems provided by acetyl substituents, plant cell wall degrading microorganisms have developed a host of acetyl esterases whose function is to deacetylate the polysaccharides prior to, or concomitant with, its complete hydrolysis by a consortium of exo-and endo-acting glycoside hydrolases. Such microbial esterases have, unsurprisingly, found widespread industrial application in both biomass conversion and for the chemoenzymatic synthesis of diverse esters (see for example Refs. 1 and 2 and reviewed in Ref.3).Xylan is a chemically and structurally complex plant cell wall polysaccharide, whose complete degradation requires the action of a dedicated enzymatic consortium, . Thus far, structures of ferulate and acetylxylan esterases have revealed a ␣/␤ hydrolase fold, and they display a Ser-His-Asp catalytic triad. Examples include the family CE1 (predominantly bacterial) ferulate esterases (8 -10), the currently unclassified fungal ferulate esterases (11-13) and the xylan and xylooligosaccharide esterases from families CE5 (14, 15), CE6 (putative esterases, PDB code 2APJ, Centre for Eukaryotic Structural Genomics), and CE7 (16). Enzymes in the largest sequence-based esterase family, CE4, do not, however, display the standard ␣/␤ hydrolase fold.The carbohydrate esterase family CE4 contains over 870 open reading frames.3 This family is notable, not merely for its size, but also as many CE4 members have been reported to be metal ion-dependent. Furthermore, family CE4 contains members with both classical de-Oacetylase activity, such as the acetylxylan est...

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-D D-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.