title: A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase.
abstract: Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Department of Chemistry, The Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Ludwiczek
authors.forename: Markus
authors.initials: M
authors.lastname: Heller
authors.forename: Terrence
authors.initials: T
authors.lastname: Kantner
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry* / Q000378:metabolism*