2WNN

Structure of wild type E. coli N-acetylneuraminic acid lyase in complex with pyruvate in space group P21

2WNN の概要

• エントリーDOI: 10.2210/pdb2wnn/pdb
• 関連するPDBエントリー: 1FDY 1FDZ 1HL2 1NAL 2WKJ 2WNQ 2WNZ 2WO5 2WPB
• 分子名称: N-ACETYLNEURAMINATE LYASE, SODIUM ION, PENTAETHYLENE GLYCOL, ... (4 entities in total)
• 機能のキーワード: carbohydrate metabolism, lyase
• 由来する生物種: ESCHERICHIA COLI
• 細胞内の位置: Cytoplasm: P0A6L4
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 135123.24

構造登録者

Campeotto, I.,Bolt, A.H.,Harman, T.A.,Trinh, C.H.,Dennis, C.A.,Phillips, S.E.V.,Pearson, A.R.,Nelson, A.,Berry, A. (登録日: 2009-07-13, 公開日: 2010-08-25, 最終更新日: 2023-12-13)

主引用文献

Campeotto, I.,Bolt, A.H.,Harman, T.A.,Dennis, C.A.,Trinh, C.H.,Phillips, S.E.V.,Nelson, A.,Pearson, A.R.,Berry, A.
Structural Insights Into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution.
J.Mol.Biol., 404:56-, 2010

PubMed 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.

PubMed: 20826162
DOI: 10.1016/J.JMB.2010.08.008

実験手法

X-RAY DIFFRACTION (1.65 Å)