3IIO

Evolutionary optimization of computationally designed enzymes: Kemp eliminases of the KE07 series

3IIO の概要

• エントリーDOI: 10.2210/pdb3iio/pdb
• 関連するPDBエントリー: 3IIP 3IIV
• 分子名称: KE07 (2 entities in total)
• 機能のキーワード: beta barrel, lyase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 174324.88

構造登録者

Khersonsky, O.,Dym, O.,Tawfik, D.S. (登録日: 2009-08-03, 公開日: 2010-03-02, 最終更新日: 2023-11-01)

主引用文献

Khersonsky, O.,Rothlisberger, D.,Dym, O.,Albeck, S.,Jackson, C.J.,Baker, D.,Tawfik, D.S.
Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series.
J.Mol.Biol., 396:1025-1042, 2010

PubMed Abstract: Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K(M) value of approximately 2600 s(-1) M(-1) and an approximately 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.

PubMed: 20036254
DOI: 10.1016/j.jmb.2009.12.031

実験手法

X-RAY DIFFRACTION (2.25 Å)