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Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

DOI: 10.1016/j.jmb.2010.08.008 DOI Help
PMID: 20826162 PMID Help

Authors: Ivan Campeotto (University of Leeds) , Amanda H. Bolt (University of Leeds) , Thomas Harman (University of Leeds) , Caitriona Dennis (University of Leeds) , Chi Hung Trinh (Institute of Molecular and Cellular Biology, University of Leeds) , Simon Phillips (Diamond Light Source) , Adam Nelson (University of Leeds) , Alan Berry (University of Leeds) , Arwen Pearson (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Molecular Biology

State: Published (Approved)
Published: September 2010

Open Access Open Access

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 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 P21 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.

Journal Keywords: Crystallography; X-Ray; Directed; Escherichia; Models; Molecular; Mutant; Mutation; Missense; Oxo-Acid-Lyases; Protein; Tertiary; Substrate Specificity

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-Macromolecular Crystallography