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

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

doi:10.1074/jbc.m800936200

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 degrees 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 approximately 25 degrees 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.

Highlights

Ing, that there have been extensive studies on the structural features of thermostable enzymes that confer resistance to inactivation at elevated temperatures
This approach, when combined with GeneReassemblyTM technology, which enables the best combination of single amino acid mutations to be incorporated into a single polypeptide by stochastic recombination of the fittest mutants identified through gene site saturation mutagenesis (GSSM), can increase enzyme thermostability Ͼ20 °C leading to the generation of hyperthermophilic variants [12,13,14]
In this report we have utilized GSSM technology to generate a hyperthermophilic variant of the catalytic module of a glycoside hydrolase family (GH) 11 [15] xylanase

Summary

Introduction

Ing, that there have been extensive studies on the structural features of thermostable enzymes that confer resistance to inactivation at elevated temperatures (see Refs. 1–3 for review). The observed increases in stability, are again relatively modest (3– 8 °C), and this methodology only examines a small proportion of the amino acid sequence space as single base changes to the codons limits the number of possible changes at each position in the primary sequence These studies mainly focus on the conversion of mesophilic enzymes, with temperature stabilities ranging from 40 to 60 °C, into thermophilic biocatalysts that are stable between 60 and 70 °C. Comparison of the crystal structure of EvXyn and EvXyn11TS showed that only one of the seven amino acid changes significantly increased direct interactions within the protein, whereas the other mutations mediated substantial improvements in stability through very subtle changes to the structure of the enzyme These data demonstrate the power of comprehensive molecular forced evolution, compared with rational design, in converting a thermostable enzyme into a hyperthermophilic variant and point to the requirement for new tools to interrogate protein folding at nonambient temperatures

Methods

Results

Conclusion