| The overall goal of this thesis was to study the Cellulomonas fimi family 10 beta-1,4-xylanase Cex along its retaining double-displacement reaction pathway using NMR spectroscopy, complemented by X-ray crystallography and enzyme kinetics.;Cex is a modular enzyme composed of a xylan-specific catalytic domain with a cellulose-binding domain, joined by a proline-threonine linker. Using NMR spectroscopy, it was demonstrated that the domains do not measurably interact with each other, and that the proline-threonine linker tethering them is predominantly unstructured and conformationally flexible on the sub-ns timescale. This supports a "beads-on-a-string" model of Cex in which it is anchored to cellulose via its cellulose-binding domain, yet cleaving nearby xylan by virtue of the flexible glycosylated linker.;Substrate binding and catalysis by Cex are controlled by an intricate network of charges and hydrogen bonding interactions within its active site. Using a plethora of isotopic labelling strategies and NMR-monitored pH titrations, the charge states and/or pKa values of all the ionizable groups (Lys, His, Glu, and Asp) in the Cex active site were determined in both its free and inhibited forms. Most importantly, the pKa of the general acid/base residue Glul27 was shown to cycle to serve its role as both a general acid (pKa 7.4) and a general base (pKa 4.2) in the glycosylation and the deglycosylation steps of the beta-retaining double-displacement reaction, respectively.;The family 10 xylanases display significant topological variations in the number of binding cleft substrate recognition sites. Two xylotriose-based inhibitors of Cex were examined by X-ray crystallography and enzyme inactivation kinetics, and these studies revealed that unlike other family 10 xylanases, Cex does not contain a significant -3 glycone binding subsite.;NMR relaxation measurements demonstrated that the backbone and tryptophan sidechains of Cex are well ordered on the ns-ps and ms-mus timescales in both its free and glycosyl-enzyme intermediate forms. However, the local and global stability of Cex increased upon glycosyl-enzyme intermediate formation. The relatively rigid active site may be necessary to bind, distort, and subsequently hydrolyze target glycosides, as well as to maintain the lifespan of the secreted xylanase in the extracellular milieu.;Together, this research provides a unique perspective of the synergistic interactions between dynamics, electrostatics, and global architecture that govern the function of the multidomain xylanase Cex. |
