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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 mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI‐TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4‐O‐methyl‐d‐glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear β‐1,4‐xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA3Xyl3 (aldotetraouronic acid), MeGlcA3Xyl4 (aldopentaouronic acid) and MeGlcA3Xyl5 (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA2Xyl2, MeGlcA2Xyl3 and MeGlcA2Xyl4. As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3–14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with β‐xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA2Xyl2. In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.

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A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA‐2Xyl‐4Xyl‐4Xyl, HexA3Xyl3), releasing the reducing‐end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo‐β‐1,4‐xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan‐hydrolyzing endo‐β‐1,4‐glucanase EG I. XYN IV was able to degrade several different β‐1,4‐xylans, but was inactive on β‐1,4‐mannans and β‐1,4‐glucans. It showed both exo‐and endo‐xylanase activity. Rhodymenan, a linear soluble β‐1,3‐β‐1,4‐xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward β‐xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4‐O‐methyl‐d‐glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

Expression of a Trichoderma reesei gene coding for a putative GH30 xylanase in Aspergillus oryzae led to isolation and purification of a novel xylanase exhibiting catalytic properties different from those of the previously characterized GH30 xylanase XYN IV of T. reesei. The novel enzyme, named XYN VI, exhibited catalytic properties similar to appendage‐dependent GH30 glucuronoxylanases previously recognized only in bacteria. XYN VI showed high specific activity only on xylans or xylooligosaccharides containing 4‐O‐methyl‐d‐glucuronic acid or d‐glucuronic acid side substituents. The cleavage of the main chain takes place primarily at the second glycosidic linkage from the branch towards the reducing end of the polysaccharides or aldouronic acids. These catalytic properties resemble bacterial GH30 glucuronoxylanases, although the recognition of the uronic acid side chains by XYN VI is apparently based on interaction of the substrate with other amino acids. Moreover, in contrast to bacterial enzymes, XYN VI is also capable of slower but significant cleavage of unsubstituted parts of xylan and acidic xylooligosaccharides. The data point to a great catalytic diversity of xylanases produced by the most extensively studied cellulolytic fungus.

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