The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain.
Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose. Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A. The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules.
Charnock
S J
SJ
Department of Chemistry, Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, U.K.
Bolam
D N
DN
Turkenburg
J P
JP
Gilbert
H J
HJ
Ferreira
L M
LM
Davies
G J
GJ
Fontes
C M
CM
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003013:Clostridium / Q000737:chemistry / Q000201:enzymology*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
D002241:Carbohydrates; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase