| 1. |
- Amrein, Beat Anton, et al.
(författare)
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CADEE : Computer-Aided Directed Evolution of Enzymes
- 2017
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Ingår i: IUCrJ. - 2052-2525. ; 4:1, s. 50-64
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Tidskriftsartikel (refereegranskat)abstract
- The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.
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| 2. |
- Bauer, Paul, et al.
(författare)
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Q6 : A comprehensive toolkit for empirical valence bond and related free energy calculations
- 2018
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Ingår i: SoftwareX. - : Elsevier. - 2352-7110. ; 7, s. 388-395
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Tidskriftsartikel (refereegranskat)abstract
- Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.
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| 3. |
- Duarte, Fernanda, et al.
(författare)
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Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
- 2014
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 118:16, s. 4351-4362
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Tidskriftsartikel (refereegranskat)abstract
- The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.
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| 4. |
- Hong, Nan-Sook, et al.
(författare)
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The evolution of multiple active site configurations in a designed enzyme
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.
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| 5. |
- Koenekoop, Lucien, et al.
(författare)
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The Activation Parameters of a Cold-Adapted Short Chain Dehydrogenase Are Insensitive to Enzyme Oligomerization
- 2022
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 61:7, s. 514-522
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Tidskriftsartikel (refereegranskat)abstract
- The structural principles of enzyme cold adaptation are of fundamental interest both for understanding protein evolution and for biotechnological applications. It has become clear in recent years that structural flexibility plays a major role in tuning enzyme activity at low temperatures, which is reflected by characteristic changes in the thermodynamic activation parameters for psychrophilic enzymes, compared to those of mesophilic and thermophilic ones. Hence, increased flexibility of the enzyme surface has been shown to lead to a lower enthalpy and a more negative entropy of activation, which leads to higher activity in the cold. This immediately raises the question of how enzyme oligomerization affects the temperature dependence of catalysis. Here, we address this issue by computer simulations of the catalytic reaction of a cold-adapted bacterial short chain dehydrogenase in different oligomeric states. Reaction free energy profiles are calculated at different temperatures for the tetrameric, dimeric, and monomeric states of the enzyme, and activation parameters are obtained from the corresponding computational Arrhenius plots. The results show that the activation free energy, enthalpy, and entropy are remarkably insensitive to the oligomeric state, leading to the conclusion that assembly of the subunit interfaces does not compromise cold adaptation, even though the mobilities of interfacial residues are indeed affected.
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| 6. |
- Machado, Teresa F. G., et al.
(författare)
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Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:24, s. 15019-15032
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD(+):acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.
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| 7. |
- Machado, Teresa F. G., et al.
(författare)
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Transition States for Psychrophilic and Mesophilic (R)-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer a Sub-zero Temperatures
- 2021
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 60:27, s. 2186-2194
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Tidskriftsartikel (refereegranskat)abstract
- (R)-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 degrees C show a much higher steady-state rate constant (k(cat)) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants (k(STO)) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on k(cat) ((D)k(cat)) and k(STO) ((D)k(STO)) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH (D)k(cat) and (D)k(STO) are generally temperature-independent. The (D)k(STO) data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 degrees C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 degrees C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 degrees C, the His-to-Asn mutation in psychrophilic HBDH decreases (D)k(cat) to 3.1, suggesting a decrease in transition-state symmetry, while the Histo-Asn mutation in mesophilic HBDH increases (D)k(cat) to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.
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| 8. |
- Oanca, Gabriel, et al.
(författare)
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Insights into enzyme point mutation effect by molecular simulation : phenylethylamine oxidation catalyzed by monoamine oxidase A
- 2016
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 18:19, s. 13346-13356
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Tidskriftsartikel (refereegranskat)abstract
- The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the alpha-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier.
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| 9. |
- Poberznik, Matic, et al.
(författare)
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Empirical Valence Bond Simulations of the Hydride-Transfer Step in the Monoamine Oxidase A Catalyzed Metabolism of Noradrenaline
- 2016
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 120:44, s. 11419-11427
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Tidskriftsartikel (refereegranskat)abstract
- Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines, such as dopamine, serotonin, and noradrenaline (NA), which is why they have been extensively implicated in the etiology and course of various neurodegenerative disorders and, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. The precise chemical mechanism through which MAOs regulate the amine Concentration, which is vital for the development of novel inhibitors, is still not unambiguously determined in the literature. In this work, we present atomistic empirical valence bond simulations of the rate-limiting step of the MAO-A-catalyzed NA (norepinephrine) degradation, involving hydride transfer from the substrate alpha-methylene group to the flavin moiety of the flavin adenine dinucleotide prosthetic group, employing the full dimensionality and thermal fluctuations of the hydrated enzyme, with extensive configurational sampling. We show that MAO-A lowers the free energy of activation by 14.3 kcal mol(-1) relative to that of the same reaction in aqueous solution, whereas the calculated activation free energy of Delta G(double dagger) = 20.3 +/- 1.6 kcal mori is found to be in reasonable agreement with the correlated experimental value of 16.5 kcal mol(-1). The results presented here strongly support the fact that both MAO-A and MAO-B isoforms function by the same hydride-transfer mechanism. We also considered a few point mutations of the "aromatic cage" tyrosine residue (Tyr444Phe, Tyr444Leu, Tyr444Trp, Tyr444His, and Tyr444Glu), and the calculated changes in the reaction barriers are in agreement with the experimental values, thus providing further support to the proposed mechanism.
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| 10. |
- Prah, Alja, et al.
(författare)
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How Monoamine Oxidase A Decomposes Serotonin : An Empirical Valence Bond Simulation of the Reactive Step
- 2020
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Ingår i: Journal of Physical Chemistry B. - : AMER CHEMICAL SOC. - 1520-6106 .- 1520-5207. ; 124:38, s. 8259-8265
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme-catalyzed degradation of the biogenic amine serotonin is an essential regulatory mechanism of its level in the human organism. In particular, monoamine oxidase A (MAO A) is an important flavoenzyme involved in the metabolism of monoamine neurotransmitters. Despite extensive research efforts, neither the catalytic nor the inhibition mechanisms of MAO enzymes are currently fully understood. In this article, we present the quantum mechanics/molecular mechanics simulation of the rate-limiting step for the serotonin decomposition, which consists of hydride transfer from the serotonin methylene group to the N5 atom of the flavin moiety. Free-energy profiles of the reaction were computed by the empirical valence bond method. Apart from the enzymatic environment, the reference reaction in the gas phase was also simulated, facilitating the estimation of the catalytic effect of the enzyme. The calculated barrier for the enzyme-catalyzed reaction of 14.82 +/- 0.81 kcal mol(-1) is in good agreement with the experimental value of 16.0 kcal mol(-1), which provides strong evidence for the validity of the proposed hydride-transfer mechanism. Together with additional experimental and computational work, the results presented herein contribute to a deeper understanding of the catalytic mechanism of MAO A and flavoenzymes in general, and in the long run, they should pave the way toward applications in neuropsychiatry.
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