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

Abstract : 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.
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Submitted on : Tuesday, July 30, 2019 - 3:29:00 PM
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Mohamed Mroueh, Marion Aruanno, Romain Borne, Pascale de Philip, Henri-Pierre Fierobe, et al.. The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action. Biotechnology for Biofuels, BioMed Central, 2019, 12 (1), ⟨10.1186/s13068-019-1483-y⟩. ⟨hal-02197666⟩

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