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

Dumon, Claire and Varvak, Alexander and Wall, Mark A. and Flint, James E. and Lewis, Richard J. and Lakey, Jeremy H. and Morland, Carl and Luginbuhl, Peter and Healey, Shaun and Todaro, Thomas and DeSantis, Grace and Sun, May and Parra-Gessert, Lilian and Tan, Xuqiu and Weiner, David P. and Gilbert, Harry J. (2008) Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. The Journal of biological chemistry, 283 (33). pp. 22557-22564. ISSN 0021-9258

Full content URL: http://dx.doi.org/10.1074/jbc.M800936200

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Abstract

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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Keywords:Amines, Amino acids, Animal cell culture, Enzymes, Flow interactions, Hydrophobicity, Industrial applications, Melting point, Organic acids, Ambient temperatures, Amino acid substitutions, Animal feeds, Catalytic properties, Combinatorial libraries, Elevated temperatures, Hydrophobic interactions, Hyperthermostability, Hyperthermostable, Industrial uses, Intramolecular interactions, Melting temperatures, Molecular bases, New tools, Paper pulps, Primary structures, Protein evolutions, Protein structures, Single mutants, Structural bases, Surface loops, Target proteins, Terminal regions, Xylanase, Xylanases, Protein folding, phenylalanine, proline, protein evxyn11, protein evxyn11 ts, serine, threonine, unclassified drug, xylan endo 1,3 beta xylosidase, endo 1,4 beta xylanase, primer DNA, amino acid sequence, amino acid substitution, amino terminal sequence, article, catalysis, crystal structure, enzyme activity, enzyme conformation, methodology, nucleotide sequence, priority journal, protein engineering, thermostability, chemistry, codon, drug stability, gene library, genetics, polymerase chain reaction, thermodynamics, Animalia, Codon, DNA Primers, Endo-1,4-beta Xylanases, Thermodynamics
Subjects:B Subjects allied to Medicine > B100 Anatomy, Physiology and Pathology
C Biological Sciences > C100 Biology
Divisions:College of Science > School of Life Sciences
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ID Code:12465
Deposited On:29 Oct 2013 09:50

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