| 1. |
- Auman, Dirk, et al.
(author)
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Peroxy Intermediate Drives Carbon Bond Activation in the Dioxygenase AsqJ
- 2022
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:34, s. 15622-15632
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Journal article (peer-reviewed)abstract
- Dioxygenases catalyze stereoselective oxygen atom transfer in metabolic pathways of biological, industrial, and pharmaceutical importance, but their precise chemical principles remain controversial. The α-ketoglutarate (αKG)-dependent dioxygenase AsqJ synthesizes biomedically active quinolone alkaloids via desaturation and subsequent epoxidation of a carbon–carbon bond in the cyclopeptin substrate. Here, we combine high-resolution X-ray crystallography with enzyme engineering, quantum-classical (QM/MM) simulations, and biochemical assays to describe a peroxidic intermediate that bridges the substrate and active site metal ion in AsqJ. Homolytic cleavage of this moiety during substrate epoxidation generates an activated high-valent ferryl (FeIV = O) species that mediates the next catalytic cycle, possibly without the consumption of the metabolically valuable αKG cosubstrate. Our combined findings provide an important understanding of chemical bond activation principles in complex enzymatic reaction networks and molecular mechanisms of dioxygenases.
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| 2. |
- Baumgart, Mona, et al.
(author)
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Design of buried charged networks in artificial proteins
- 2021
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Journal article (peer-reviewed)abstract
- Soluble proteins are universally packed with a hydrophobic core and a polar surface that drive the protein folding process. Yet charged networks within the central protein core are often indispensable for the biological function. Here, we show that natural buried ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif from its surroundings to gain structural stability. To explore this effect, we build artificial proteins with buried ion-pairs by combining directed computational design and biophysical experiments. Our findings illustrate how perturbation in charged networks can introduce structural rearrangements to compensate for desolvation effects. We validate the physical principles by resolving high-resolution atomic structures of the artificial proteins that are resistant towards unfolding at extreme temperatures and harsh chemical conditions. Our findings provide a molecular understanding of functional charged networks and how point mutations may alter the protein's conformational landscape.
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| 3. |
- Bräuer, Alois, et al.
(author)
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Structural snapshots of the minimal PKS system responsible for octaketide biosynthesis
- 2020
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In: Nature Chemistry. - : Springer Science and Business Media LLC. - 1755-4330 .- 1755-4349. ; 12:8
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Journal article (peer-reviewed)abstract
- The invariable core of a type II polyketide synthase has been characterized using X-ray crystallography, simulations, mutagenesis experiments and functional assays. The characterization of the ternary acyl carrier protein complexes provides a mechanistic understanding of the reactivity and could inform future engineering of this complex biosynthetic machinery. Type II polyketide synthases (PKSs) are multi-enzyme complexes that produce secondary metabolites of medical relevance. Chemical backbones of such polyketides are produced by minimal PKS systems that consist of a malonyl transacylase, an acyl carrier protein and an alpha/beta heterodimeric ketosynthase. Here, we present X-ray structures of all ternary complexes that constitute the minimal PKS system for anthraquinone biosynthesis inPhotorhabdus luminescens. In addition, we characterize this invariable core using molecular simulations, mutagenesis experiments and functional assays. We show that malonylation of the acyl carrier protein is accompanied by major structural rearrangements in the transacylase. Principles of an ongoing chain elongation are derived from the ternary complex with a hexaketide covalently linking the heterodimeric ketosynthase with the acyl carrier protein. Our results for the minimal PKS system provide mechanistic understanding of PKSs and a fundamental basis for engineering PKS pathways for future applications.
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| 4. |
- Di Trani, Justin M., et al.
(author)
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Structural basis of mammalian complex IV inhibition by steroids
- 2022
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:30
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Journal article (peer-reviewed)abstract
- The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.
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| 5. |
- Jussupow, Alexander, et al.
(author)
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Extended conformational states dominate the Hsp90 chaperone dynamics
- 2022
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In: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 298:7
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Journal article (peer-reviewed)abstract
- The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a beta-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.
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| 6. |
- Mader, Sophie L., et al.
(author)
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Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90
- 2020
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Journal article (peer-reviewed)abstract
- The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.
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| 7. |
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| 8. |
- Parmesan, C, et al.
(author)
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Poleward shifts in geographical ranges of butterfly species associated with regional warming
- 1999
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In: NATURE. - : MACMILLAN MAGAZINES LTD. - 0028-0836. ; 399:6736, s. 579-583
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Journal article (other academic/artistic)abstract
- Mean global temperatures have risen this century, and further warming is predicted to continue for the next 50-100 years(1-3) Some migratory species can respond rapidly to yearly climate variation by altering the timing or destination of migration(4), but
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| 9. |
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| 10. |
- Raff, Elizabeth C., et al.
(author)
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Doushantuo fossils are not giant bacteria, but bacterial pseudomorphs of animal embryos
- 2008
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In: The Palaeontological Association.
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Conference paper (peer-reviewed)abstract
- Embryos from the Ediacaran Doushantuo Formation are among the most astonishing examples of exceptional fossilization. However, the mechanism of fossilization ispoorly understood, leading directly to debate over the interpretation of the fossils, someauthors even questioning their interpretation as embryos. It has been hypothesized thatmicrobial processes are responsible for preservation and mineralization of organic tissues.However, the actions of microbes in preservation of embryos have not been demonstratedexperimentally. We show that bacterial biofilms assemble rapidly in marine embryos,forming detailed pseudomorphs of cellular organization and structure. We define threeessential steps in embryo preservation: 1) blockage of autolysis by reducing or anaerobic conditions; 2) rapid formation of microbial biofilms that consume the embryo butform a replica that retains cell organization and morphology; 3) bacterially-catalyzedmineralization. We identified major bacterial taxa in embryo decay biofilms using16S rDNA sequencing. Decay processes were similar in different taphonomic conditions,but bacterial populations depended on specific conditions. Experimental taphonomyresembles preservation states of fossils. Our data show how fossilization of soft tissues insediments is mediated by bacterial replacement and mineralization, providing a foundationfor experimentally creating biofilms from defined microbial species to model fossilization asa biological process.
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