The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis

- The crystal structure of a thermostable quadruple GH11 xylanase mutant was determined.
- Molecular dynamics simulations were performed for the mutant and wild-type enzymes.
- The structural data were correlated with thermodynamics analyses for all mutants.
- GH11 thermostability is due to backbone rigidity, solvation and hydrophobic contacts.

A thermostable variant of the mesophilic xylanase A from Bacillus subtilis (BsXynA-G3_4x) contains the four mutations Gln7His, Gly13Arg, Ser22Pro, and Ser179Cys. The crystal structure of the BsXynA-G3_4x has been solved, and the local environments around each of these positions investigated by molecular dynamics (MD) simulations at 328 K and 348 K. The structural and MD simulation results were correlated with thermodynamic data of the wild-type enzyme, the 4 single mutants and the BsXynA-G3_4x. This analysis suggests that the overall stabilizing effect is entropic, and is consistent with solvation of charged residues and reduction of main-chain flexibility. Furthermore, increased protein-protein hydrogen bonding and hydrophobic interactions also contribute to stabilize the BsXynA-G3_4x. The study revealed that a combination of several factors is responsible for increased thermostability of the BsXynA-G3_4x; (i) introduction of backbone rigidity in regions of high flexibility, (ii) solvation effects and (iii) hydrophobic contacts.

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