Functional and structural characterization of AtAbf43C: an exo-1,5-α-L-arabinofuranosidase from Acetivibrio thermocellus DSM1313

Abstract

Acetivibrio thermocellus degrades diverse polysaccharides found in plant biomass using an array of glycoside hydrolase (GH) enzymes. Here, we describe the structure and function of AtAbf43C, an uncharacterized GH family 43 subfamily 26 (GH43_26) α-L-arabinofuranosidase (EC 3.2.1.55) from A. thermocellus. AtAbf43C is optimally active on para-nitrophenol-α-L-arabinofuranoside at pH 5.5 and 65 °C, making it the most thermophilic bacterial GH43_26 enzyme characterized to date. We solved high-resolution crystal structures of full-length AtAbf43C and its individual carbohydrate binding module family 42 (CBM42) and GH43 domains, including a structure with L-arabinofuranose molecules bound to the CBM42. The CBM42 domain adopts a typical β-trefoil fold, and the GH43 domain forms a canonical 5-bladed β-propeller, each resembling those in the mesophilic GH43_26 enzyme SaAraf43A from Streptomyces avermitilis (PDB 3AKH). However, AtAbf43C exhibits a unique domain organization, with the CBM42 at the N-terminus and the GH43 domain at the C-terminus, the reverse of the arrangement observed in SaAraf43A. Structural alignment enabled identification of the conserved catalytic triad (D168, D283, and E344) in AtAbf43C, which we confirmed experimentally with site-directed mutagenesis. The deep-narrow topology of the AtAbf43C GH43 binding pocket is consistent with exo activity on arabino-oligosaccharide (AOS) substrates. Indeed, liquid chromatography-mass spectrometry (LC-MS) analysis of polysaccharides and oligosaccharides hydrolyzed by AtAbf43C confirmed exo activity primarily toward α-1,5-linked AOSs. This suggests AtAbf43C contributes to the degradation of AOS released from arabinose-rich polysaccharides by other A. thermocellus enzymes. Together, these results expand our understanding of the structure-function of GH43_26 enzymes and their role in plant biomass deconstruction.

Keywords: Acetivibrio thermocellus; Clostridium thermocellum; arabino-oligosaccharides; carbohydrate binding protein; glycoside hydrolase; α-L-arabinofuranosidase.

Figure 1. Sequence analysis and purification of AtAbf43C.

(A) The general architecture of the Clo1313_2794 Gene. (B) A phylogenetic tree constructed after a multiple protein sequence alignment using ClustalOmega of the GenBank protein accession # (ADU75776.1) corresponding to Clo1313_2794 (★) with the other characterized members of the GH43_26 subfamily. (C) Protein gel of the purified full-length AtAbf43C protein and its truncated versions: AtAbf43C_CBM42 and AtAbf43C_GH43. CBM, carbohydrate binding module; GH, glycoside hydrolase.

Figure 2. Effect of environmental conditions on AtAbf43C activity on pNPAra.

(A) pH optimization performed at 55 °C. (B) Temperature optimization performed at pH 5.5. (C) Thermostability test of AtAbf43C. (D) Effect of various 10 mM additives on enzymatic activity. Asterisks indicate statistical significance (P≤0.05) compared with the untreated condition. Error bars represent standard deviations between triplicate technical replicates at each reaction condition. pNPAra, Para-nitrophenol-α-l-arabinofuranoside.

Figure 3. The major structural features of AtAbf43C shown using the arabinose-soaked structure (PDB code: 9NXH).

Magnesium (Mg²⁺) is shown as a purple sphere, glycerol molecules are shown as green stick structures, and arabinose molecules are shown as dark gray stick structures. (a) The overall structure of AtAbf43C with the GH43 domain shown in yellow and the CBM42 domain shown in red. (b) The five-bladed β-propeller structure of the GH43 domain, with each blade shown in a distinct color. (c) The three catalytic residues labeled and shown as stick structures within the GH43 structure. (d) The β-trefoil structure of the CBM42 domain, with each subdomain shown in a distinct color. (e) Arabinose bound in the β-pocket of the CBM42 domain. (f) Arabinose bound in the γ-pocket of the CBM42 domain. Contacted residues in (e & f) are labeled and shown as stick structures. Hydrogen bonds between the residues and arabinose molecule bonds are shown as dashed cyan lines, with bond lengths labeled in Å. CBM, carbohydrate binding module; GH, glycoside hydrolase.

Figure 4. Structural comparison of AtAbf43C and SaAraf43A.

(a) Structural alignment of the GH43 domain of arabinose soaked AtAbf43C structure (GH43 domain in yellow, CBM42 domain in red) with the structure of SaAraf43A complexed with arabinotriose (PDB Code: 3AKH) shown in light blue. (b) Alignment of the catalytic residues in SaAraf43A (light blue) and co-crystallized arabinose and arabinobiose molecules in SaAraf43A structure with the active site residues of AtAbf43C (yellow). (c) Superimposition of the arabinose and arabinobiose molecules from the SaAraf43A structure onto the surface structure of AtAbf43C, with the AtAbf43C catalytic residues shown in yellow. In (a-c), sodium (Na⁺) is shown as an orange sphere, chlorine (Cl^-) is shown as a green sphere, glycerol molecules are shown as green stick structures, and sugar molecules are shown as dark gray stick structures. CBM, carbohydrate binding module; GH, glycoside hydrolase.