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- Linder, Mats, 1983-, et al.
(författare)
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Computational design of a Diels-Alderase from a thermophilic esterase : the importance of dynamics
- 2012
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; 26:9, s. 1079-1095
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 2. |
- Linder, Mats, et al.
(författare)
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Computational design of a lipase for catalysis of the Diels-Alder reaction
- 2011
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Ingår i: Journal of Molecular Modeling. - : Springer Science and Business Media LLC. - 1610-2940 .- 0948-5023. ; 17:4, s. 833-849
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Tidskriftsartikel (refereegranskat)abstract
- Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like nearattack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the alpha,beta-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates (v (cat) /v (uncat) ) of up to 103 are predicted.
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| 3. |
- Linder, Mats, et al.
(författare)
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Designing a New Diels-Alderase : A Combinatorial, Semirational Approach Including Dynamic Optimization
- 2011
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Ingår i: JOURNAL OF CHEMICAL INFORMATION AND MODELING. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 51:8, s. 1906-1917
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Tidskriftsartikel (refereegranskat)abstract
- A computationally inexpensive design strategy involving 'semirational' screening for enzymatic catalysis is presented. The protocol is based on well-established computational methods and represents a holistic approach to the catalytic process. The model reaction studied here is the Diels-Alder, for which a successful computational design has recently been published (Siegel, J. B. et al. Science 2010, 329, 309-313). While it is a leap forward in the field of computational design, the focus on designing only a small fraction of the active site gives little control over dynamics. Our approach explicitly incorporates mutagenesis and the analysis of binding events and transition states, and a promising enzyme substrate candidate is generated with relatively little effort. We estimate catalytic rate accelerations of up to 10(5).
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