The xyl - doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α- l -arabinofuranosidases with complementary modes of action

Background The α- l -arabinofuranosidases (α- l -ABFs) are exoenzymes involved in the hydrolysis of α- l -arabinosyl linkages in plant cell wall polysaccharides. They play a crucial role in the degradation of arabinoxylan and arabinan and they are used in many biotechnological applications. Analysis of the genome of R. cellulolyticum showed that putative cellulosomal α- l -ABFs are exclusively encoded by the xyl - doc gene cluster, a large 32-kb gene cluster. Indeed, among the 14 Xyl-Doc enzymes encoded by this gene cluster, 6 are predicted to be α- l -ABFs belonging to the CAZyme families GH43 and GH62. Results The biochemical characterization of these six Xyl-Doc enzymes revealed that four of them are α- l -ABFs. GH43 16 -1229 (RcAbf43A) which belongs to the subfamily 16 of the GH43, encoded by the gene at locus Ccel_1229, has a low specific activity on natural substrates and can cleave off arabinose decorations located at arabinoxylan chain extremities. GH43 10 -1233 (RcAbf43A d2,3 ), the product of the gene at locus Ccel_1233, belonging to subfamily 10 of the GH43, can convert the double arabinose decorations present on arabinoxylan into single O2- or O3-linked decorations with high velocity ( k cat = 16.6 ± 0.6 s −1 ). This enzyme acts in synergy with GH62-1234 (RcAbf62A m2,3 ), the product of the gene at locus Ccel_1234, a GH62 α- l -ABF which hydrolyzes α-(1 → 3) or α-(1 → 2)-arabinosyl linkages present on polysaccharides and arabinoxylooligosaccharides monodecorated. Finally, a bifunctional enzyme, GH62-CE6-1240 (RcAbf62B m2,3 Axe6), encoded by the gene at locus Ccel_1240, which contains a GH62-α- l -ABF module and a carbohydrate esterase (CE6) module, catalyzes deacylation of plant cell wall polymers and cleavage of arabinosyl mono-substitutions. These enzymes are also active on arabinan, a component of the type I rhamnogalacturonan, showing their involvement in pectin degradation. Conclusion Arabinofuranosyl decorations on arabinoxylan and pectin strongly inhibit the action of xylan-degrading enzymes and pectinases. α- l -ABFs encoded by the xyl - doc gene cluster of R. cellulolyticum can remove all the decorations present in the backbone of arabinoxylan and arabinan, act synergistically, and, thus, play a crucial role in the degradation of plant cell wall polysaccharides.