Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl‐enzyme intermediate

Abstract

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

“…The H/D exchange patterns for the studied structures are indicative of an enzyme whose backbone is well ordered (Figs. S4 and S5), in agreement with the previous NMR measurements (35).…”

A Secondary Xylan-binding Site Enhances the Catalytic Activity of a Single-domain Family 11 Glycoside Hydrolase

“…Previous NMR spectroscopic and X-ray crystallographic analyses of wild-type (WT) and 2FX2-BcX demonstrated that only active site residues are perturbed structurally, dynamically, and spectroscopically due to the covalent attachment of this inhibitor to Glu78. 22,36 Upon titration of 2FX2-BcX with X4, residues within the SBS exhibited similar chemical shift perturbations in the fast exchange limit as seen with the E78Q-BcX variant, while the blocked AS residues remained completely unaffected ( Figure 2). To perform the complementary experiment, we disrupted the putative SBS by substituting alanine for three residues, N141, N181, and T183, which showed large main chain 1 H N -15 N and side-chain 1 H δ -15 N δ chemical shift perturbations in the presence of X4 (Supplementary Data Figure S1).…”

“…22,45 15 N relaxation studies also demonstrated that the active site of BcX is rigid on the nanosecond to picosecond timescale in both its apo and covalently modified forms. 36 Thus, this conformational change may reflect millisecond to microsecond timescale motions of the thumb region necessary for substrate binding and release. Alternatively, whereas both xylose residues in the −2 and −1 subsites of a noncovalent complex of X4 with inactive E172C-BcX maintained 4 C 1 chair conformations, 23 the covalently linked proximal xylose in WT 2FX2-BcX was found to be distorted into a 2,5 B boat conformation.…”

“…The spectral assignments of WT-BcX and 2FX2-BcX have been reported. 24,36 Along with consideration of these data, the main-chain 1 H-15 N signals of 0.8 mM 13 C/ 15 N-labeled E78Q-BcX, in 10 mM sodium phosphate (pH 6.0) and 10% (v/v) 2 H 2 O were assigned from sensitivity-enhanced 1 H-15 N HSQC, HNCACB, and CBCACONH spectra recorded at 30°C. 57 Side-chain asparagine 1 H δ2 -15 N δ2 and glutamine 1 H ε2 -15 N ε2 peaks were identified from HNCACB and C(CO)TOCSY-NH experiments optimized for NH 2 groups.…”

Circular Permutation of Bacillus circulans Xylanase: A Kinetic and Structural Study

“…The other (Glu172) functions as a general acid/base catalyst, assisting in the formation of the intermediate by proton donation, as well as in the subsequent hydrolysis step by deprotonating the attacking nucleophilic water. The active site residues of Bcx, as well as the dynamic and electrostatic properties of the glycosylenzyme intermediate, have been characterized extensively by X-ray crystallography and NMR spectroscopy (5,(7)(8)(9)(10)(11)(12).Studies of Bcx, along with parallel investigations on another xylanase of a different tertiary fold (the GH family 10 Cellulomonas fimi endoxylanase Cex) and continuing collaborative computational and heavy atom isotope effect studies, have provided particularly detailed insights into fundamental mechanisms of carbohydrate degradation (6,13,14). With this same set of mechanistic tools, analyses have been performed on interesting mutants derived from site-directed and random mutagenesis methods, thereby providing additional insights into structure/ function relationships.…”