Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose.

Agarose is a gel-forming polysaccharide with an alpha-L(1,4)-3,6-anhydro-galactose, beta-D(1,3)-galactose repeat unit, from the cell walls of marine red algae. beta-agarase A, from the Gram-negative bacterium Zobellia galactanivorans, is secreted to the external medium and degrades agarose with an endo-mechanism. The structure of the inactive mutant beta-agarase A-E147S in complex with agaro-octaose has been solved at 1.7 A resolution. Two oligosaccharide chains are bound to the protein. The first one resides in the active site channel, spanning subsites -4 to -1. A second oligosaccharide binding site, on the opposite side of the protein, was filled with eight sugar units, parallel to the active site. The crystal structure of the beta-agarase A with agaro-octaose provides detailed information on agarose recognition in the catalytic site. The presence of the second, parallel, binding site suggests that the enzyme might be able to unwind the double-helical structure of agarose prior to the catalytic cleavage.