β-1,3-Glucan Binding by a Thermostable Carbohydrate-Binding Module from Thermotoga maritima

• Published in: Biochemistry (Easton) 2001-12, Vol.40 (48), p.14679-14685
• Main Authors: Boraston, Alisdair B, Warren, R. Antony J, Kilburn, Douglas G
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; beta-Glucans; Binding, Competitive; Chromatography, Affinity; DNA Primers - chemistry; Glucan Endo-1,3-beta-D-Glucosidase - isolation & purification; Glucans - metabolism; Hot Temperature; Mutagenesis, Site-Directed; Osmosis; Plasmids; Polymerase Chain Reaction; Polysaccharides - isolation & purification; Polysaccharides - metabolism; Protein Binding; Substrate Specificity; Thermotoga maritima - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi015760g

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T. maritima comprise a highly thermostable family 4 CBM that binds β-1,3- and β-(1,3)(1,4)-glucans. Laminarin, a β-1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 × 107 M-1) and one with a 100-fold lower affinity (2.4 × 105 M-1). The affinities for laminarioligosaccharides and β-(1,3)(1,4)-glucans ranged from ∼2 × 105 to ∼2.5 × 106 M-1. Cellooligosaccharides and laminariobiose were bound only very weakly (K as ∼5 × 103 M-1). Spectroscopic and mutagenic studies implicated the involvement of three tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with large negative changes in heat capacity. Temperature and osmotic conditions profoundly influenced binding. For the first time in solution, the direct uptake and release of water in CBM binding are demonstrated.