Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.

2.50
Hdl Handle:
http://hdl.handle.net/11287/619055
Title:
Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.
Authors:
Campeotto, I.; Bolt, Amanda H. ( 0000-0003-4303-2718 ) ; Harman, T. A.; Dennis, C.; Trinh, C. H.; Phillips, S. E. V.; Nelson, A.; Pearson, A. R.; Berry, A.
Abstract:
The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2(1) at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.
Citation:
Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution. 2010, 404 (1):56-69 J. Mol. Biol.
Publisher:
Elsevier
Journal:
Journal of molecular biology
Issue Date:
19-Nov-2010
URI:
http://hdl.handle.net/11287/619055
DOI:
10.1016/j.jmb.2010.08.008
PubMed ID:
20826162
Additional Links:
http://www.sciencedirect.com/science/article/pii/S0022283610008570
Note:
This article is freely available via Open Access. Click on the 'Additional Link' above to access the full-text from the publisher's site.
Type:
Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
Language:
en
ISSN:
1089-8638
Appears in Collections:
pre-2014 RD&E publications

Full metadata record

DC FieldValue Language
dc.contributor.authorCampeotto, I.en
dc.contributor.authorBolt, Amanda H.en
dc.contributor.authorHarman, T. A.en
dc.contributor.authorDennis, C.en
dc.contributor.authorTrinh, C. H.en
dc.contributor.authorPhillips, S. E. V.en
dc.contributor.authorNelson, A.en
dc.contributor.authorPearson, A. R.en
dc.contributor.authorBerry, A.en
dc.date.accessioned2016-08-30T09:51:47Z-
dc.date.available2016-08-30T09:51:47Z-
dc.date.issued2010-11-19-
dc.identifier.citationStructural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution. 2010, 404 (1):56-69 J. Mol. Biol.en
dc.identifier.issn1089-8638-
dc.identifier.pmid20826162-
dc.identifier.doi10.1016/j.jmb.2010.08.008-
dc.identifier.urihttp://hdl.handle.net/11287/619055-
dc.description.abstractThe substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2(1) at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.en
dc.language.isoenen
dc.publisherElsevieren
dc.relation.urlhttp://www.sciencedirect.com/science/article/pii/S0022283610008570en
dc.rightsArchived with thanks to Journal of molecular biologyen
dc.subjectWessex Classification Subject Headings::Biochemistryen
dc.titleStructural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.en
dc.typeResearch Support, N.I.H., Extramuralen
dc.typeResearch Support, Non-U.S. Gov'ten
dc.identifier.journalJournal of molecular biologyen
dc.description.noteThis article is freely available via Open Access. Click on the 'Additional Link' above to access the full-text from the publisher's site.en
dc.description.fundingDK064265/DK/NIDDK NIH HHS/United Statesen
dc.type.versionPublisheden

Related articles on PubMed

All Items in RD&E Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.