Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Brinck)
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Brinck)
Cambridge Crystallographic Data Centre.
Cambridge Crystallographic Data Centre.
Show others and affiliations
2012 (English)In: Journal of Computer-Aided Molecular Design, ISSN 0920-654X, E-ISSN 1573-4951, Vol. 26, no 9, 1079-1095 p.Article in journal (Refereed) Published
Abstract [en]

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).

Place, publisher, year, edition, pages
2012. Vol. 26, no 9, 1079-1095 p.
Keyword [en]
Diels-Alder, Computational enzyme design, DFT, Molecular dynamics
National Category
Theoretical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-101711DOI: 10.1007/s10822-012-9601-yISI: 000310089500009Scopus ID: 2-s2.0-84869507791OAI: oai:DiVA.org:kth-101711DiVA: diva2:548773
Funder
Swedish Research Council
Note

QC 20121206

Available from: 2012-08-31 Created: 2012-08-31 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Computational Studies and Design of Biomolecular Diels-Alder Catalysis
Open this publication in new window or tab >>Computational Studies and Design of Biomolecular Diels-Alder Catalysis
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Diels-Alder reaction is one of the most powerful synthetic tools in organic chemistry, and asymmetric Diels-Alder catalysis allows for rapid construction of chiral carbon scaffolds. For this reason, considerable effort has been invested in developing efficient and stereoselective organo- and biocatalysts. However, Diels-Alder is a virtually unknown reaction in Nature, and to engineer an enzyme into a Diels-Alderase is therefore a challenging task. Despite several successful designs of catalytic antibodies since the 1980’s, their catalytic activities have remained low, and no true artificial ’Diels-Alderase’ enzyme was reported before 2010.

In this thesis, we employ state-of-the-art computational tools to study the mechanism of organocatalyzed Diels-Alder in detail, and to redesign existing enzymes into intermolecular Diels-Alder catalysts. Papers I–IV explore the mechanistic variations when employing increasingly activated reactants and the effect of catalysis. In particular, the relation between the traditionally presumed concerted mechanism and a stepwise pathway, forming one bond at a time, is probed. Papers V–X deal with enzyme design and the computational aspects of predicting catalytic activity. Four novel, computationally designed Diels-Alderase candidates are presented in Papers VI–IX. In Paper X, a new parameterization of the Linear Interaction Energy model for predicting protein-ligand affinities is presented.

A general finding in this thesis is that it is difficult to attain large transition state stabilization effects solely by hydrogen bond catalysis. In addition, water (the preferred solvent of enzymes) is well-known for catalyzing Diels- Alder by itself. Therefore, an efficient Diels-Alderase must rely on large binding affinities for the two substrates and preferential binding conformations close to the transition state geometry. In Papers VI–VIII, we co-designed the enzyme active site and substrates in order to achieve the best possible complementarity and maximize binding affinity and pre-organization. Even so, catalysis is limited by the maximum possible stabilization offered by hydrogen bonds, and by the inherently large energy barrier associated with the [4+2] cycloaddition.

The stepwise Diels-Alder pathway, proceeding via a zwitterionic intermediate, may offer a productive alternative for enzyme catalysis, since an enzyme active site may be more differentiated towards stabilizing the high-energy states than for the standard mechanism. In Papers I and III, it is demonstrated that a hydrogen bond donor catalyst provides more stabilization of transition states having pronounced charge-transfer character, which shifts the preference towards a stepwise mechanism.

Another alternative, explored in Paper IX, is to use an α,β -unsaturated ketone as a ’pro-diene’, and let the enzyme generate the diene in situ by general acid/base catalysis. The results show that the potential reduction in the reaction barrier with such a mechanism is much larger than for conventional Diels-Alder. Moreover, an acid/base-mediated pathway is a better mimic of how natural enzymes function, since remarkably few catalyze their reactions solely by non-covalent interactions.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. xii, 138 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2012:34
Keyword
Computational chemistry, density functional theory, enzyme design, molecular modeling, organocatalysis, stepwise Diels-Alder, oxyanion hole
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-101706 (URN)978-91-7501-435-7 (ISBN)
Public defence
2012-09-21, K1, Teknikringen 56, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20120903

Available from: 2012-09-03 Created: 2012-08-31 Last updated: 2012-09-03Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Authority records BETA

Brinck, Tore

Search in DiVA

By author/editor
Linder, MatsJohansson, Adam JohannesBrinck, Tore
By organisation
Applied Physical Chemistry
In the same journal
Journal of Computer-Aided Molecular Design
Theoretical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 124 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf