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Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain

Item Type:Article
Title:Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain
Creators Name:Ay, J. and Goetz, F. and Borriss, R. and Heinemann, U.
Abstract:The 1,3-1,4-beta-glucanase from Bacillus macerans (wtGLU) and the 1, 4-beta-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 A resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.
Keywords:Bacillus, Endo-1,4-beta Xylanases, Glycoside Hydrolases, Plasmids, Protein Folding, Recombinant Fusion Proteins, Structure-Activity Relationship, Xylosidases
Source:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
Publisher:National Academy of Sciences (U.S.A.)
Volume:95
Number:12
Page Range:6613-6618
Date:9 June 1998
Official Publication:http://www.pnas.org/content/95/12/6613.abstract
PubMed:View item in PubMed

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