Strategies for Modulating the pH-Dependent Activity of a Family 11 Glycoside Hydrolase

The pH-dependent activity of wild-type <i>Bacillus circulans</i> xylanase (BcX) is set by the p<i>K</i>_a values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these p<i>K</i>_a values and thereby shift the pH_opt at which BcX is optimally active. Altering the global charge of BcX through random succinylation had no significant effect. Mutation of residues near or within the active site of BcX, but not directly contacting the catalytic carboxyls, either had little effect or reduced its pH_opt, primarily by lowering the apparent p<i>K</i>_a value of Glu78. However, mutations causing the largest p<i>K</i>_a changes also impaired activity. Although not found as a general acid/base in naturally occurring xylanases, substitution of Glu172 with a His lowered the pH_opt of BcX from 5.6 to 4.7 while retaining 8% activity toward a xylobioside substrate. Mutation of Asn35, which contacts Glu172, to either His or Glu also led to a reduction in pH_opt by ∼1.2 units. Detailed p<i>K</i>_a measurements by NMR spectroscopy revealed that, despite the opposite charges of the introduced residues, both the N35H and N35E forms of BcX utilize a reverse protonation mechanism. In this mechanism, the p<i>K</i>_a value of the general acid is lower than that of the nucleophile, and only a small population of enzyme is in a catalytically competent ionization state. However, overall activity is maintained due to the increased strength of the general acid. This study illustrates several routes for altering the pH-dependent properties of xylanases, while also providing valuable insights into complex protein electrostatics

Results from the NMR-driven restrained molecular dynamics simulation of oxoG4 and oxoG10.

<p>(A) The difference in backbone conformation between BI and BII conformations. ε and ζ are highlighted in accordance to their torsion angle definition, as BI and BII are defined by the ε–ζ. For both DDD BI and oxoG4 BII structures, the G₄ and A₅ are shown and rotated to highlight the backbone differences. The DDD BI conformation (left panel) is from the PDB ID INAJ structure. The BII conformation induced by oxoG4 (right panel) is from our averaged minimized structure, PDB ID 5IV1. Carbons are shown in black, oxygen is shown in red, nitrogen in blue and hydrogens in white. The additional oxygen at C8 and hydrogen at N7 in the oxoG₄ base are color-coded in the same way. (B) The ε–ζ shows BII directly 3' of the modification site for oxoG4 and oxoG10. BI is defined as ε–ζ less than 20°, with over 20° defined as BII. The nucleotide sequence numbers correspond to the steps in the sequence, with ε–ζ for nucleotide step 1 corresponding to the torsion angles between C₁ and G₂ in C₁G₂C₃G₄A₅A₆T₇T₈C₉G₁₀C₁₁G₁₂. In black with open square markers, are the ε–ζ for 1NAJ (DDD). In red with closed square markers are ε–ζ for oxoG4 and blue with closed circle markers are oxoG10. (C) oxoG4 and oxoG10 cause significant unwinding near the modification site. Color schemes and data origins are the same as in panel B.</p

Kinetics of EcoRI on oxoG-containing substrates.

<p>Kinetics of EcoRI on oxoG-containing substrates.</p

Cleavage of native and oxoG-modified GAATTC sequence by <i>Eco</i>RI.

<p>Odd lanes, enzyme added (300 pM); even lanes, no enzyme (controls). The concentration of the substrate was 100 nM. The central part of the 30-mer duplexes and the position of oxoG (X) and radioactive label (asterisk) are shown below the image. Cleavage of the top strand produces a 12-mer product; cleavage of the bottom strand produces a 14-mer product.</p