beta-1,3-Glucan binding by a thermostable carbohydrate-binding module from Thermotoga 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 approximately 2 x 10(5) to approximately 2.5 x 10(6) M(-1). Cellooligosaccharides and laminariobiose were bound only very weakly (K(a)s approximately 5 x 10(3) 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.
Boraston
A B
AB
Protein Engineering Network of Centres of Excellence, Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada. boraston@ysbl.york.ac.uk
Warren
R A
RA
Kilburn
D G
DG
D020816:Amino Acid Motifs; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000302:isolation & purification; D005936:Glucans / Q000378:metabolism*; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D009995:Osmosis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000737:chemistry*; D047071:beta-Glucans
D017931:DNA Primers; D005936:Glucans; D011134:Polysaccharides; D047071:beta-Glucans; C008247:laminaran; C033363:beta-1,3-glucan; D004693:Glucan Endo-1,3-beta-D-Glucosidase