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Engineering hyperthermostability into a GH
xylanase is mediated by subtle changes to protein structure
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Dr Claire Dumon
Dr James Flint
Professor Rick Lewis
Professor Jeremy Lakey
Professor Harry Gilbert
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
Journal of Biological Chemistry
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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,
Xyn11. 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 (
) 1–8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant,
, containing seven mutations.
∼ 25 °C higher than the parent enzyme while displaying catalytic properties that were similar to
Xyn11. The crystal structures of
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.
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