5EHT

Indirect contributions of mutations underlie optimization of new enzyme function

5EHT の概要

• エントリーDOI: 10.2210/pdb5eht/pdb
• 関連するPDBエントリー: 5EH9
• 分子名称: N-acyl homoserine lactonase, ZINC ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: n-acyl-homoserine lactonase, directed evolution, aiia, qql, lactonase, phosphatase, paraoxonase, hydrolase
• 由来する生物種: Bacillus thuringiensis
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29151.71

構造登録者

Jackson, C.J.,Hong, N.-S. (登録日: 2015-10-28, 公開日: 2016-09-07, 最終更新日: 2024-10-30)

主引用文献

Yang, G.,Hong, N.,Baier, F.,Jackson, C.J.,Tokuriki, N.
Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.
Biochemistry, 55:4583-4593, 2016

PubMed Abstract: How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.

PubMed: 27444875
DOI: 10.1021/acs.biochem.6b00561

実験手法

X-RAY DIFFRACTION (1.29 Å)