Abstract
The Acetivibrio thermocellus DSM1313 genome codes for seven predicted glycoside hydrolase family 43 (GH43) enzymes, four of which remain uncharacterized. This study describes the function and structure of one such enzyme, AtAbf43C, from GH43 subfamily 26 (GH43_26) which acts as an α-L-arabinofuranosidase (EC 3.2.1.55). AtAbf43C is active on para-nitrophenol-α-L-arabinofuranoside (pNPAra), with optimal activity observed at pH 5.5 and 65 ℃. Multiple crystal structures of AtAbf43C were obtained, in which an N-terminal carbohydrate binding module family 42 (CBM42) domain displays a β-trefoil type fold and the C-terminal GH43 domain displays a canonical 5-bladed β-propeller motif. One structure, which was solved with two L-arabinofuranose molecules bound to β- and γ-subdomains of the CBM42, builds upon previous literature suggesting the α-binding pocket of the AtAbf43C CBM42 is non-functional. Furthermore, structural alignment with the substrate bound structure of a closely homologous GH43_26 exo-α-1,5-arabinofuranosidase, SaAraf43A from Streptomyces avermitilis (PDB 3AKH), allowed for identification of the conserved catalytic triad via site-directed mutagenesis in AtAbf43C, as well as insight into the deep-narrow topology of the AtAbf43C binding pocket that suggested it would be active on similar arabino-oligosaccharide (AOS) substrates as SaAraf43A. Subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of polysaccharides and oligosaccharides hydrolyzed by AtAbf43C provides experimental evidence confirming this enzyme acts in an exo manner primarily towards α-1,5 linked arabino-oligosaccharides.