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An exo-␤-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-␤-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward ␤-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze ␤-1,4-linked galacto-oligosaccharides, ␤-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze ␤-1,3-galactosyl galactosaminide, ␤-1,3-galactosyl glucosaminide, or ␤-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal ␤-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from ␤-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl ␤-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass ␤-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and ␤-1,3-glucan, although it bound ␤-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a ␤-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.Arabinogalactan proteins (AGPs) 1 are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (1, 2). Although their precise functions are unknown, AGPs have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (3-5). AGPs are characterized by large amounts of carbohydrate components rich in galactose (sugars in the present study are D series unless otherwise designated) and Larabinose, and protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (1). The carbohydrate moieties of AGPs have a common structure consisting of a ␤-1,3-galactosyl backbone to which side chains of ␤-1,6-galactosyl residues are attached through O-6. L-Arabinose and lesser amounts of other auxiliary sugars, such as glucuronic acid, L-rhamnose, and L-fucose, are attached to t...

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Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific ␣-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave ␣-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific ␣-1,2-arabinofuranosidase, CjAbf43A, displays a fivebladed ␤-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for ␣-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

Background: Glycoside hydrolase family 62 ␣-L-arabinofuranosidases specifically release L-arabinose from arabinoxylan. Results: The crystal structure of glycoside hydrolase family 62 ␣-L-arabinofuranosidase from Streptomyces coelicolor was determined. Conclusion: L-Arabinose and xylohexaose complexed structures revealed the mechanism of substrate specificity of this enzyme. Significance: Efficient catalysis by glycoside hydrolase family 62 ␣-L-arabinofuranosidase requires its binding to terminal xylose sugars at the substrate-binding cleft.

Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a ␤-Larabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) ␣-D-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-␤-L-arabinopyranoside was 18 mol of arabinose/min/mg, which was 67 times higher than that toward pnitrophenyl-␣-D-galactopyranoside. The enzyme could remove 0.1 and 45% L-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel ␤-domain containing Greek key motifs, another antiparallel ␤-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of ␣-D-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of ␤-L-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of D-galactose bound to ␣-galactosidase was changed in ␤-L-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which ␤-L-arabinopyranosidase is classified as a new member of the GH27 family.Arabinogalactan proteins (AGPs) 3 are a family of complex proteoglycans widely distributed in plants (1, 2). AGPs are also found in tree exudate gums and coniferous woods (3) and are characterized by the presence of large amounts of carbohydrate components rich in galactose (all the sugars in the present study are in the D-configuration unless otherwise specified) and L-arabinose and by protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (4). Type II arabinogalactans and short oligosaccharides are the two types of carbohydrates attached to the AGP backbone. Type II arabinogalactans have ␤-1,3-linked galactosyl backbones in mono-or oligo-␤-1,6-galactosyl and/or L-arabinosyl side chains (2, 5). L-Arabinose and lesser amounts of other auxiliary sugars such as glucuronic acid, L-rhamnose, and L-fucose are attached to the side chains primarily at nonreducing termini (2). Molecular and biochemical evidence indicates that AGPs have specific functions during root formation, promotion of somatic embryogenesis, and attraction of pollen tubes to the style (6). However, because many putative protein cores exist and the structures of the carbohydrate moieties are complex, it has been difficult...

A gene encoding an exo-␤-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-␤-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of ␤-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from ␤-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a ␤-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-␤-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including ␤-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a ␤-1,3-galactobiose, ␤-1,4-galactosyl glucose, or ␤-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward ␤-1,3-galactan and ␣-Larabinofuranosidase-treated AGP.

Background:The three-dimensional structures of ␤-glucuronidase have been solved only for the GH2 enzymes. Results: AcGlcA79A is composed of a (␤/␣) 8 -barrel domain and a ␤-domain. Conclusion:The substrate binding site of AcGlcA79A is adapted for recognition of GlcA as a substrate. Significance: This is the first report describing the crystal structure, mechanism, and catalytic residues of a GH79 enzyme.