1H6Y

The role of conserved amino acids in the cleft of the C-terminal family 22 carbohydrate binding module of Clostridium thermocellum Xyn10B in ligand binding

Summary for 1H6Y

• Entry DOI: 10.2210/pdb1h6y/pdb
• Related: 1DYO 1H6X
• Descriptor: ENDO-1,4-BETA-XYLANASE Y, CALCIUM ION (3 entities in total)
• Functional Keywords: xylan degradation, hydrolase, glycosidase
• Biological source: CLOSTRIDIUM THERMOCELLUM
• Total number of polymer chains: 2
• Total formula weight: 37959.85

Authors

Xie, H.,Bolam, D.N.,Charnock, S.J.,Davies, G.J.,Williamson, M.P.,Simpson, P.J.,Fontes, C.M.G.A.,Ferreira, L.M.A.,Gilbert, H.J. (deposition date: 2001-06-29, release date: 2002-06-27, Last modification date: 2023-12-13)

Primary citation

Xie, H.,Gilbert, H.J.,Charnock, S.J.,Davies, G.J.,Williamson, M.P.,Simpson, P.J.,Raghothama, S.,Fontes, C.M.G.A.,Dias, F.M.,Ferreira, L.M.A.,Bolam, D.N.
Clostridium Thermocellum Xyn10B Carbohydrate-Binding Module 22-2: The Role of Conserved Amino Acids in Ligand Binding
Biochemistry, 40:9167-, 2001

PubMed Abstract: The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.

PubMed: 11478884
DOI: 10.1021/BI0106742

Experimental method

X-RAY DIFFRACTION (2.2 Å)