期刊
JOURNAL OF COMPUTER-AIDED MOLECULAR DESIGN
卷 26, 期 9, 页码 1079-1095出版社
SPRINGER
DOI: 10.1007/s10822-012-9601-y
关键词
Diels-Alder; Computational enzyme design; DFT; Molecular dynamics
资金
- Swedish Research Council (VR)
- Cambridge Crystallographic Data Centre (CCDC)
A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of a parts per thousand 10(3)-10(6) and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, 'ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme (T (opt) = 70 A degrees C).
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据