Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding.
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.
Xie
H
H
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
Gilbert
H J
HJ
Charnock
S J
SJ
Davies
G J
GJ
Williamson
M P
MP
Simpson
P J
PJ
Raghothama
S
S
Fontes
C M
CM
Dias
F M
FM
Ferreira
L M
LM
Bolam
D N
DN
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D050260:Carbohydrate Metabolism; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D008024:Ligands; D009844:Oligosaccharides; D014990:Xylans; D014364:Tryptophan; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase