A β-l-Arabinopyranosidase from Streptomyces avermitilis Is a Novel Member of Glycoside Hydrolase Family 27

Abstract

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...

The abp gene in Geobacillus stearothermophilus T‐6 encodes a GH27 β‐l‐arabinopyranosidase

“…Most enzymes of this family exhibit a-galactosidase activity [20], however, this type of activity does not seem to correlate with the physical location of the abp gene in the context of the arabinan operon. Recently, enzymes from family GH27 have been shown to exhibit b-L-arabinopyranosidase activity [21,22]. These findings promoted us to test the activity of the abp gene product.…”

Molecular Cloning and Characterization of a β-l-Arabinobiosidase in Bifidobacterium longum That Belongs to a Novel Glycoside Hydrolase Family

“…In addition, glycoside hydrolases in the GH42 and GH27 families possess ␣-and ␤-L-arabinopyranosidase activities, respectively (20,21). In this study, we cloned and characterized a hypothetical protein, HypBA2, and identified it as a novel ␤-Larabinobiosidase.…”

Functional Analysis of Degradative Enzymes for Hydroxyproline-linked ^|^beta;-L-Arabinofuranosides in Bifidobacterium longum

“…Glycoside hydrolases involved in the degradation of α-Larabinofuranosides are present in 6 glycoside hydrolase (GH) families (GH3, GH43, GH51, GH54, GH62, and GH93) in the CAZy database. In addition, degradative enzymes for α-and β-L-arabinopyranoside linkages were also found in the GH42 and GH27 families, respectively (1,2). On the other hand, β-L-arabinofuranoside linkages constitute sugar chains of hydroxyproline-rich glycoproteins (HRGPs), which are known as extensin and Solanaceae lectins.…”