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 enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze ␤-mannans and ␤-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 ␤-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (؊1) and two aglycone (؉1 and ؉2) sugarbinding subsites. The ؊1 subsite displays absolute specificity for mannose, whereas the ؉1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The ؉2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 Å crystal structure reveals that CmMan5A adopts a (␤/␣) 8 barrel fold, and superimposition with GH5 endomannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand ␤8 and helix ␣8 comprising residues 378 -412. This insertion forms a "double" steric barrier, formed by two short ␤-strands that function to "block" the substrate binding cleft at the edge of the ؊1 subsite forming the "exo" active center topology of CmMan5A.The plant cell wall represents the major source of organic carbon within the biosphere. The polysaccharides contained within these composite structures are hydrolyzed by an extensive repertoire of glycoside hydrolases, and the sugars released are then utilized as carbon and energy sources by a range of organisms. Mannose-containing polysaccharides are an important component of the plant cell wall and are present mainly as galactomannans in which the ␤1,4-linked mannopyranoside backbone is decorated with galactosyl residues at the O-6 position and glucomannan that contains a heterogeneous backbone of ␤1,4-linked glucose and mannose sugars (1) (Fig. 1). The polymeric backbone of these polysaccharides is hydrolyzed by endo-␤-1,4-mannanases (hereafter "mannanase" (2), whereas the side chains of galactomannan are removed by ␣-galactosidases (3). The mannooligosaccharides generated by these enzymes are further hydrolyzed by ␤-mannosidases (EC 3.2.1.25), which are exo-acting glycoside hydrolases that catalyze the removal of the nonreducing end ␤-D-mannose (2). In the sequence-based classification of glycoside hydrolases (4, 5) 1 endo-mannanases are found in families GH5 2 and GH26 with ␤-mannosidases located in families GH1 and GH2.Although mannanases have been extensively characterized, the precise catalytic profile of...

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Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.