beta-1,3-glucan binding by a thermostable carbohydrate-binding module fromThermotoga maritima

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T . maritima comprise a highly thermostable family 4 CBM that binds beta -1,3 - and beta-(1,3)(1,4)-glucans. Laminarin, a beta -1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 x 10(7) M-1) and one with a 100-fold lower affinity (2.4 x 10(5) M-1). The affinities for laminarioligosaccharides and beta-(1,3)(1,4)-glucans ranged from similar to2 x 10(5) to similar to2.5 x 10(6) M-1. Cellooligosaccharide s and laminariobiose were bound only very weakly (K(a)s similar to5 x 10(3) M-1). Spectroscopic and mutagenic studies implicated the involvement of th ree tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with lar ge negative changes in heat capacity. Temperature and osmotic conditions pr ofoundly influenced binding. For the first time in solution, the direct upt ake and release of water in CBM binding are demonstrated.