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J. Biol. Chem., Vol. 283, Issue 33, 22557-22564, August 15, 2008

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Engineering Hyperthermostability into a GH11 Xylanase Is Mediated by Subtle Changes to Protein Structure^*

Claire Dumon¹, Alexander Varvak¹, Mark A. Wall, James E. Flint, Richard J. Lewis, Jeremy H. Lakey, Carl Morland, Peter Luginbühl, Shaun Healey, Thomas Todaro, Grace DeSantis, May Sun, Lilian Parra-Gessert, Xuqiu Tan, David P. Weiner², and Harry J. Gilbert³

From the Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle Upon Tyne NE2 4HH, United Kingdom and the Verenium Corporation, San Diego, California 92121

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^TM (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 (T_m) 1–8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11^TS, containing seven mutations. EvXyn11^TS had a T_m 25 °C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11^TS 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.

Received for publication, February 5, 2008 , and in revised form, May 30, 2008.

The atomic coordinates and structure factors (codes 2VUJ and 2VUL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed. E-mail: david.weiner{at}verenium.com. ³ To whom correspondence may be addressed. E-mail: h.j.gilbert{at}ncl.ac.uk.

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