title: Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A.
abstract: Glycoside hydrolases often contain multiple copies of noncatalytic carbohydrate binding modules (CBMs) from the same or different families. Currently, the functional importance of this complex molecular architecture is unclear. To investigate the role of multiple CBMs in plant cell wall hydrolases, we have determined the polysaccharide binding properties of wild type and various derivatives of Cellulomonas fimi xylanase 11A (Cf Xyn11A). This protein, which binds to both cellulose and xylan, contains two family 2b CBMs that exhibit 70% sequence identity, one internal (CBM2b-1), which has previously been shown to bind specifically to xylan and the other at the C-terminus (CBM2b-2). Biochemical characterization of CBM2b-2 showed that the module bound to insoluble and soluble oat spelt xylan and xylohexaose with K(a) values of 5.6 x 10(4), 1.2 x 10(4), and 4.8 x 10(3) M(-1), respectively, but exhibited extremely weak affinity for cellohexaose (<10(2) M(-1)), and its interaction with insoluble cellulose was too weak to quantify. The CBM did not interact with soluble forms of other plant cell wall polysaccharides. The three-dimensional structure of CBM2b-2 was determined by NMR spectroscopy. The module has a twisted "beta-sandwich" architecture, and the two surface exposed tryptophans, Trp 570 and Trp 602, which are in a perpendicular orientation with each other, were shown to be essential for ligand binding. In addition, changing Arg 573 to glycine altered the polysaccharide binding specificity of the module from xylan to cellulose. These data demonstrate that the biochemical properties and tertiary structure of CBM2b-2 and CBM2b-1 are extremely similar. When CBM2b-1 and CBM2b-2 were incorporated into a single polypeptide chain, either in the full-length enzyme or an artificial construct comprising both CBM2bs covalently joined via a flexible linker, there was an approximate 18-20-fold increase in the affinity of the protein for soluble and insoluble xylan, as compared to the individual modules, and a measurable interaction with insoluble acid-swollen cellulose, although the K(a) (approximately 6.0 x 10(4) M(-1)) was still much lower than for insoluble xylan (K(a) = approximately 1.0 x 10(6) M(-1)). These data demonstrate that the two family 2b CBMs of Cf Xyn11A act in synergy to bind acid swollen cellulose and xylan. We propose that the increased affinity of glycoside hydrolases for polysaccharides, through the synergistic interactions of CBMs, provides an explanation for the duplication of CBMs from the same family in some prokaryotic cellulases and xylanases.
authors.affiliation: Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
authors.forename: D N
authors.initials: DN
authors.lastname: Bolam
authors.forename: H
authors.initials: H
authors.lastname: Xie
authors.forename: P
authors.initials: P
authors.lastname: White
authors.forename: P J
authors.initials: PJ
authors.lastname: Simpson
authors.forename: S M
authors.initials: SM
authors.lastname: Hancock
authors.forename: M P
authors.initials: MP
authors.lastname: Williamson
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002482:Cellulose / Q000378:metabolism; D004357:Drug Synergism; D043364:Endo-1,4-beta Xylanases; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D011134:Polysaccharides; D014990:Xylans; D014364:Tryptophan; D002482:Cellulose; D001120:Arginine; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D043364:Endo-1,4-beta Xylanases