3NPV

Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution

3NPV の概要

• エントリーDOI: 10.2210/pdb3npv/pdb
• 関連するPDBエントリー: 3NPU 3NPW 3NPX 3NQ2 3NQ8 3NQV 3NR0
• 分子名称: deoxyribose phosphate aldolase (2 entities in total)
• 機能のキーワード: tim, structural genomics, israel structural proteomics center, ispc, lyase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 55887.71

構造登録者

Khersonsky, O.,Rothlisberge, D.,Wollacott, A.M.,Dym, O.,Baker, D.,Tawfik, D.S.,Israel Structural Proteomics Center (ISPC) (登録日: 2010-06-29, 公開日: 2011-02-09, 最終更新日: 2023-11-01)

主引用文献

Khersonsky, O.,Rothlisberger, D.,Wollacott, A.M.,Murphy, P.,Dym, O.,Albeck, S.,Kiss, G.,Houk, K.N.,Baker, D.,Tawfik, D.S.
Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution
J.Mol.Biol., 407:391-412, 2011

PubMed Abstract: Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5×10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.

PubMed: 21277311
DOI: 10.1016/j.jmb.2011.01.041

実験手法

X-RAY DIFFRACTION (1.48 Å)