Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases.
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 pKa 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 (Tm) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and Tm, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pKa values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pKa values significantly lower than standard values.
Kongsted
Jacob
J
Department of Theoretical Chemistry, Chemical Center, University of Lund, S221 00 Lund, Sweden.
Ryde
Ulf
U
Wydra
James
J
Jensen
Jan H
JH
D001230:Aspergillus / Q000201:enzymology / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000378:metabolism; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D013696:Temperature
D043364:Endo-1,4-beta Xylanases