title: Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase.
abstract: Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA'), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.
authors.affiliation: Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, U.K.
authors.forename: L M
authors.initials: LM
authors.lastname: Ferreira
authors.forename: A J
authors.initials: AJ
authors.lastname: Durrant
authors.forename: J
authors.initials: J
authors.lastname: Hall
authors.forename: G P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002482:Cellulose / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014990:Xylans; D002482:Cellulose; D000469:Alkaline Phosphatase; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase