Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution
| Autor: | Chi H. Trinh, Alan Berry, Arwen R. Pearson, Adam Nelson, Thomas Harman, Amanda H. Bolt, Ivan Campeotto, Simon E. V. Phillips, Caitriona Dennis |
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| Rok vydání: | 2010 |
| Předmět: |
Models
Molecular ManNAc N-acetylmannosamine Stereochemistry NAL N-acetylneuraminic acid lyase substrate specificity Protein Data Bank (RCSB PDB) Mutation Missense Biology 010402 general chemistry medicine.disease_cause Crystallography X-Ray DPAH (5R 6R)-7-(dipropylamino)-4 5 6-trihydroxy-2 7-dioxoheptanoic acid 01 natural sciences Article 03 medical and health sciences Structural Biology PDB Protein Data Bank medicine directed evolution Escherichia coli Molecular Biology 030304 developmental biology PEG polyethylene glycol X-ray crystallography chemistry.chemical_classification 0303 health sciences Escherichia coli Proteins Substrate (chemistry) Oxo-Acid-Lyases DHOB (2R 3S)-2 3-dihydroxy-4-oxo-N N-dipropylbutanamide protein engineering Protein engineering THB (2R 3R)-2 3 4-trihydroxy-N N-dipropylbutanamide Directed evolution Lyase Neu5Ac N-acetylneuraminic acid 0104 chemical sciences Protein Structure Tertiary N-acetylneuraminic acid lyase Enzyme chemistry Biochemistry Amino Acid Substitution Aldol condensation Mutant Proteins Directed Molecular Evolution |
| Zdroj: | Journal of Molecular Biology |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2010.08.008 |
| Popis: | 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 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. |
| Databáze: | OpenAIRE |
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