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Title: Engineering glycoside hydrolase stability by the introduction of zinc binding

Abstract

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

Authors:
; ; ; ORCiD logo; ; ; ; ; ; ;
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1457487
Alternate Identifier(s):
OSTI ID: 1476933; OSTI ID: 1506325
Report Number(s):
SAND-2018-9901J
Journal ID: ISSN 2059-7983; ACSDAD; PII: S2059798318006678
Grant/Contract Number:  
AC02-76SF00515; AC04-94AL85000; AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Acta Crystallographica. Section D. Structural Biology
Additional Journal Information:
Journal Name: Acta Crystallographica. Section D. Structural Biology Journal Volume: 74 Journal Issue: 7; Journal ID: ISSN 2059-7983
Publisher:
IUCr
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; glycoside hydrolases; protein engineering; thermal stability; X-ray crystallography

Citation Formats

Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., and Adams, Paul D. Engineering glycoside hydrolase stability by the introduction of zinc binding. United Kingdom: N. p., 2018. Web. doi:10.1107/S2059798318006678.
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Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., & Adams, Paul D. Engineering glycoside hydrolase stability by the introduction of zinc binding. United Kingdom. https://doi.org/10.1107/S2059798318006678
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Ellinghaus, Thomas L., Pereira, Jose H., McAndrew, Ryan P., Welner, Ditte H., DeGiovanni, Andy M., Guenther, Joel M., Tran, Huu M., Feldman, Taya, Simmons, Blake A., Sale, Kenneth L., and Adams, Paul D. Wed . "Engineering glycoside hydrolase stability by the introduction of zinc binding". United Kingdom. https://doi.org/10.1107/S2059798318006678.
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@article{osti_1457487,
title = {Engineering glycoside hydrolase stability by the introduction of zinc binding},
author = {Ellinghaus, Thomas L. and Pereira, Jose H. and McAndrew, Ryan P. and Welner, Ditte H. and DeGiovanni, Andy M. and Guenther, Joel M. and Tran, Huu M. and Feldman, Taya and Simmons, Blake A. and Sale, Kenneth L. and Adams, Paul D.},
abstractNote = {The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.},
doi = {10.1107/S2059798318006678},
journal = {Acta Crystallographica. Section D. Structural Biology},
number = 7,
volume = 74,
place = {United Kingdom},
year = {Wed Jun 27 00:00:00 EDT 2018},
month = {Wed Jun 27 00:00:00 EDT 2018}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1107/S2059798318006678
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Table 1 Table 1: Data-collection, refinement and model statistics.
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