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Endo-1,5-α-l-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-l-arabinan, releasing arabino-oligosaccharides and l-arabinose. Two extracellular endo-1,5-α-l-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-l-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-l-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-l-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.