Structural highlights
Function
XYNA_NIACI
Publication Abstract from PubMed
The pH-dependent activity of wild-type Bacillus circulans xylanase (BcX) is set by the pKa values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these pKa values and thereby shift the pHopt 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 pHopt, primarily by lowering the apparent pKa value of Glu78. However, mutations causing the largest pKa changes also impaired activity. Although not found as a general acid/base in naturally occurring xylanases, substitution of Glu172 with a His lowered the pHopt 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 pHopt by approximately 1.2 units. Detailed pKa 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 pKa 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.
Strategies for Modulating the pH-Dependent Activity of a Family 11 Glycoside Hydrolase.,Ludwiczek ML, D'Angelo I, Yalloway GN, Brockerman JA, Okon M, Nielsen JE, Strynadka NC, Withers SG, McIntosh LP Biochemistry. 2013 May 7;52(18):3138-56. doi: 10.1021/bi400034m. Epub 2013 Apr, 24. PMID:23578322[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Ludwiczek ML, D'Angelo I, Yalloway GN, Brockerman JA, Okon M, Nielsen JE, Strynadka NC, Withers SG, McIntosh LP. Strategies for Modulating the pH-Dependent Activity of a Family 11 Glycoside Hydrolase. Biochemistry. 2013 May 7;52(18):3138-56. doi: 10.1021/bi400034m. Epub 2013 Apr, 24. PMID:23578322 doi:10.1021/bi400034m
