A UNIQUE MULTI-DOMAIN EXTRACELLULAR GH43 ARABINANASE DETERMINED IN DIFFERENT CONFORMATIONAL STATES

a-L-arabinanases are key enzymes in the breakdown of arabinan, one of the main polysaccharides in the plant cell-wall, and hence present a wide range of important potential biotechnological applications. GsAbn43A is an extracellular a-L-arabinanase from the thermophilic Gram-positive bacterium Geobacillus stearothermophilus-T6, shown to degrade efficiently linear and naturally-branched arabinan. The enzyme belongs to family GH43, contains 848 amino-acid residues, and possesses relatively low sequence identity to related arabinanases. The 3D structure of GsAbn43A has recently been determined by X-ray crystallography, revealing a unique and novel multi-domain architecture, the largest reported so far in the GH43 family. The enzyme is built of four different domains, arranged in a pincer-like structure. The catalytic domain (shown in red in the figures below) corresponds to the five-bladed b-propeller fold observed in GH43 enzymes. The second domain (green) has also been seen before in some homologous two-domain arabinanases, but the third (blue) and fourth (orange) domains are unique to GsAbn43A. Interestingly, two different conformational states have been determined for the enzyme, a "closed" (right figure) and an "open" state (left), differentiated by a ~12 Å movement in location of the fourth domain. Substrate-binding structural experiments demonstrate surprisingly that an arabinopentaose substrate binds to a dedicated site in the fourth domain, rather than to the expected catalytic domain and active site (central figure). These findings, together with complementary ITC and kinetics experiments, suggest a novel catalytic mechanism for the arabinan degradation action of GsAbn43A.