Arabinogalactan-proteins Degrading Enzymes

Abstract

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans found throughout the plant kingdom. AGPs are massively glycosylated, and are implicated in diverse plant growth and development. Gum arabic and larch arabinogalactan, which are a kind of AGP, were used for food materials and additives. Carbohydrate moieties of AGPs consist of β-1,3-galactan backbone having β-1,6-galactan side-chains. Galactanases that hydrolyze β-1,3- or β-1,6-galactans are important in degrading AGPs. We succeeded in, for the first time, cloning an exo-β-1,3-galactanase and an endo-β-1,6-galactanase genes from Phanerochaete chrysosporium (Pc1,3Gal43A) and Trichoderma viride (Tv6GAL), respectively. Pc1,3Gal43A consisted of two modules resembling glycoside hydrolase family 43 (GH43) and carbohydrate binding module family 35 (CBM35). It specifically hydrolyzed only the β-1,3-linkage of two galactosyl residues in an exo-acting manner. However, it produced oligosaccharides together with galactose from AGPs, suggesting that Pc1,3Gal43A is able to accommodate β-1,6-linked galactosyl side-chains. The C-terminal CBM35 specifically bound to β-1,3-galactan. Using the Pc1,3Gal43A sequence as a query in BLAST search, we newly obtained the enzymes from Clostridium thermocellum and Streptomyces avermitilis. Both enzymes contained GH43 and CBM13. We found similar sequences not only in bacteria but also in plants. The two kinds of gene products from Arabidopsis thaliana demonstrated exo-β-1,3-galactanase activity when they were expressed in yeast. Thus, exo-β-1,3-galactanases are distributed in the fungal, bacterial and plant kingdoms. The putative endo-β-1,6-galactanase gene from S. avermitilis which was found by using Tv6GAL sequence was cloned. The recombinant enzyme catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides in an endo-acting manner.