| 1. |
- Bauer, Paul, et al.
(författare)
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Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
- 2016
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Ingår i: Organic and biomolecular chemistry. - 1477-0520 .- 1477-0539. ; 14:24, s. 5639-5651
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Tidskriftsartikel (refereegranskat)abstract
- Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.
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| 2. |
- Ben-David, Moshe, et al.
(författare)
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Catalytic Stimulation by Restrained Active-Site Floppiness-The Case of High Density Lipoprotein-Bound Serum Paraoxonase-1
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:6, s. 1359-1374
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Tidskriftsartikel (refereegranskat)abstract
- Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
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| 3. |
- Ben-David, Moshe, et al.
(författare)
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Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:4, s. 1133-1147
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Tidskriftsartikel (refereegranskat)abstract
- Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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| 4. |
- Burke, Jason R., et al.
(författare)
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Bifunctional Substrate Activation via an Arginine Residue Drives Catalysis in Chalcone Isomerases
- 2019
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435 .- 2155-5435. ; 9:9, s. 8388-8396
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Tidskriftsartikel (refereegranskat)abstract
- Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex combined with molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Bronsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.
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| 5. |
- Crean, Rory M., et al.
(författare)
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KIF-Key Interactions Finder : A program to identify the key molecular interactions that regulate protein conformational changes
- 2023
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Ingår i: Journal of Chemical Physics. - : American Institute of Physics (AIP). - 0021-9606 .- 1089-7690. ; 158:14
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Tidskriftsartikel (refereegranskat)abstract
- Simulation datasets of proteins (e.g., those generated by molecular dynamics simulations) are filled with information about how a non-covalent interaction network within a protein regulates the conformation and, thus, function of the said protein. Most proteins contain thousands of non-covalent interactions, with most of these being largely irrelevant to any single conformational change. The ability to automatically process any protein simulation dataset to identify non-covalent interactions that are strongly associated with a single, defined conformational change would be a highly valuable tool for the community. Furthermore, the insights generated from this tool could be applied to basic research, in order to improve understanding of a mechanism of action, or for protein engineering, to identify candidate mutations to improve/alter the functionality of any given protein. The open-source Python package Key Interactions Finder (KIF) enables users to identify those non-covalent interactions that are strongly associated with any conformational change of interest for any protein simulated. KIF gives the user full control to define the conformational change of interest as either a continuous variable or categorical variable, and methods from statistics or machine learning can be applied to identify and rank the interactions and residues distributed throughout the protein, which are relevant to the conformational change. Finally, KIF has been applied to three diverse model systems (protein tyrosine phosphatase 1B, the PDZ3 domain, and the KE07 series of Kemp eliminases) in order to illustrate its power to identify key features that regulate functionally important conformational dynamics.
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| 6. |
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| 7. |
- Janfalk Carlsson, Åsa, et al.
(författare)
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Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
- 2016
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Ingår i: ChemBioChem (Print). - : Wiley. - 1439-4227 .- 1439-7633. ; 17:18, s. 1693-1697
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Tidskriftsartikel (refereegranskat)abstract
- Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.
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| 8. |
- Kaltenbach, Miriam, et al.
(författare)
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Evolution of chalcone isomerase from a noncatalytic ancestor
- 2018
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Ingår i: Nature Chemical Biology. - : NATURE PUBLISHING GROUP. - 1552-4450 .- 1552-4469. ; 14:6, s. 548-555
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substratebinding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.
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| 9. |
- Kulkarni, Yashraj S., et al.
(författare)
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Role of Ligand-Driven Conformational Changes in Enzyme Catalysis : Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:11, s. 3854-3857
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Tidskriftsartikel (refereegranskat)abstract
- We have previously performed empirical valence bond calculations of the kinetic activation barriers, Delta G(calc) double dagger, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA.HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Delta G(calc)(double dagger) for the TIM-catalyzed deprotonation of GAP (12.9 +/- 0.8 kcal.mol(-1)), of the substrate piece GA (15.0 +/- 2.4 kcal.mol(-1)), and of the pieces GA.HP, (15.5 +/- 3.5 kcal.mol(-1)). The effect of bound dianion on Delta G(calc) double dagger is small (<= 2.6 kcal.mol(-1)), in comparison to the much larger 12.0 and 5.8 kcal.mol(-1) intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA. HP, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.
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| 10. |
- Longo, Liam M., et al.
(författare)
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Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 117:10, s. 5310-5318
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in alpha beta alpha sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an alpha-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first alpha beta alpha sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.
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