Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure

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  2. Dr Claire Dumon
  3. Dr James Flint
  4. Professor Rick Lewis
  5. Professor Jeremy Lakey
  6. Carl Morland
  7. Professor Harry Gilbert
Author(s)Dumon C, Varvak A, Wall MA, Flint JE, Lewis RJ, Lakey JH, Morland C, Luginbuhl P, Healey S, Todaro T, DeSantis G, Sun M, Parra-Gessert L, Tan XQ, Weiner DP, Gilbert HJ
Publication type Article
JournalJournal of Biological Chemistry
Year2008
Volume283
Issue33
Pages22557-22564
ISSN (print)0021-9258
ISSN (electronic)1083-351X
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Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis™ (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (Tm) 1–8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS, containing seven mutations. EvXyn11TS had a Tm ∼ 25 °C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc.
URLhttp://dx.doi.org/10.1074/jbc.M800936200
DOI10.1074/jbc.M800936200
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