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A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination

Smith, Matthew A. and Rentmeister, Andrea and Snow, Christopher D. and Wu, Timothy and Farrow, Mary F. and Mingardon, Florence and Arnold, Frances H. (2012) A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination. FEBS Journal, 279 (24). pp. 4453-4465. ISSN 1742-464X. https://resolver.caltech.edu/CaltechAUTHORS:20130114-102154716

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Abstract

Sequence diversity within a family of functional enzymes provides a platform for elucidating structure–function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) – Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS – to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence–property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequence–function relationships within a family of enzymes by supplementing natural diversity with synthetic diversity.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1111/febs.12032DOIArticle
http://onlinelibrary.wiley.com/doi/10.1111/febs.12032/abstractPublisherArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2012 The Authors Journal compilation. © 2012 FEBS. Received 16 June 2012; revised 10 October 2012; accepted 12 October 2012. Article first published online: 9 Nov. 2012. This work was supported by the Department of the Interior through grant D10AP00065 from the Defense Advanced Research Projects Agency to F. H. Arnold. M. A. Smith is supported by a Resnick Sustainability Institute fellowship, A. Rentmeister by a DFG postdoctoral fellowship, and T. Wu by a CIT summer undergraduate research fellowship (SURF).
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)D10AP00065
Resnick Sustainability InstituteUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Subject Keywords:cellulases; cellulosomal; Cel48; SCHEMA recombination
Issue or Number:24
Record Number:CaltechAUTHORS:20130114-102154716
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130114-102154716
Official Citation:Smith, M. A., Rentmeister, A., Snow, C. D., Wu, T., Farrow, M. F., Mingardon, F. and Arnold, F. H. (2012), A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination. FEBS Journal, 279: 4453–4465. doi: 10.1111/febs.12032
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:36350
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:14 Jan 2013 21:15
Last Modified:03 Oct 2019 04:37

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