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. GH43₁₆-1229 (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. GH43₁₀-1233 (RcAbf43Ad₂,₃), 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 (kcₐₜ = 16.6 ± 0.6 s⁻¹). This enzyme acts in synergy with GH62-1234 (RcAbf62Aₘ₂,₃), 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 (RcAbf62Bₘ₂,₃Axe6), 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.