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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.