Advanced

Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases

Kongsted, Jacob LU ; Ryde, Ulf LU ; Wydra, James and Jensen, Jan H. (2007) In Biochemistry 46(47). p.13581-13592
Abstract
This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of... (More)
This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of 2-4.6. The influence of dynamical fluctuations of the protein structure is investigated for Bacillus agaradhaerens and Aspergillus kawachii xylanase using molecular dynamics (MD) simulations to provide snapshots from which average values can be computed. This decreases the respective predicted pH optima from 6.2-6.7 and 4.8 to 5.3 +/- 0.3 and 4.0 +/- 0.2, respectively, which are in better agreement with the observed values of 5.6 and 2, respectively. The change is primarily due to structural fluctuations of an Arg residue near the catalytic nucleophile, which lowers its pK(a) value compared to using the X-ray structure. The MD simulations and some X-ray structures indicate that this Arg residue can form a hydrogen bond to the catalytic base, and it is hypothesized that this hydrogen bond is stabilized by an additional hydrogen bond to another Glu residue present only in acidic xylanases. Formation of such a hydrogen bond is predicted to lower the pH optimum of A. kawachii xylanase to 2.9 +/- 0.3, which is in reasonable agreement, with the observed value of 2. The predicted pH of optimal stability is in excellent agreement with the pH value at which the melting temperature (T-m) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and T-m, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pK(a) values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pK(a) values significantly lower than standard values. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
46
issue
47
pages
13581 - 13592
publisher
The American Chemical Society
external identifiers
  • wos:000251150000012
  • scopus:36749020127
ISSN
0006-2960
DOI
10.1021/bi7016365
language
English
LU publication?
yes
id
6e689e27-fecd-4abd-9769-2c3ff8b56783 (old id 968956)
date added to LUP
2008-01-29 15:04:07
date last changed
2017-01-01 04:56:21
@article{6e689e27-fecd-4abd-9769-2c3ff8b56783,
  abstract     = {This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of 2-4.6. The influence of dynamical fluctuations of the protein structure is investigated for Bacillus agaradhaerens and Aspergillus kawachii xylanase using molecular dynamics (MD) simulations to provide snapshots from which average values can be computed. This decreases the respective predicted pH optima from 6.2-6.7 and 4.8 to 5.3 +/- 0.3 and 4.0 +/- 0.2, respectively, which are in better agreement with the observed values of 5.6 and 2, respectively. The change is primarily due to structural fluctuations of an Arg residue near the catalytic nucleophile, which lowers its pK(a) value compared to using the X-ray structure. The MD simulations and some X-ray structures indicate that this Arg residue can form a hydrogen bond to the catalytic base, and it is hypothesized that this hydrogen bond is stabilized by an additional hydrogen bond to another Glu residue present only in acidic xylanases. Formation of such a hydrogen bond is predicted to lower the pH optimum of A. kawachii xylanase to 2.9 +/- 0.3, which is in reasonable agreement, with the observed value of 2. The predicted pH of optimal stability is in excellent agreement with the pH value at which the melting temperature (T-m) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and T-m, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pK(a) values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pK(a) values significantly lower than standard values.},
  author       = {Kongsted, Jacob and Ryde, Ulf and Wydra, James and Jensen, Jan H.},
  issn         = {0006-2960},
  language     = {eng},
  number       = {47},
  pages        = {13581--13592},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases},
  url          = {http://dx.doi.org/10.1021/bi7016365},
  volume       = {46},
  year         = {2007},
}