| 1. |
- Irani, Mehdi, et al.
(författare)
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Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:7, s. 1280-1289
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB,(M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.
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| 2. |
- Jafari, Sonia, et al.
(författare)
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Benchmark Study of Redox Potential Calculations for Iron-Sulfur Clusters in Proteins
- 2022
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; 61:16, s. 5991-6007
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Tidskriftsartikel (refereegranskat)abstract
- Redox potentials have been calculated for 12 different iron-sulfur sites of 6 different types with 1-4 iron ions. Structures were optimized with combined quantum mechanical and molecular mechanical (QM/MM) methods, and the redox potentials were calculated using the QM/MM energies, single-point QM methods in a continuum solvent or by QM/MM thermodynamic cycle perturbations. We show that the best results are obtained with a large QM system (∼300 atoms, but a smaller QM system, ∼150 atoms, can be used for the QM/MM geometry optimization) and a large value of the dielectric constant (80). For absolute redox potentials, the B3LYP density functional method gives better results than TPSS, and the results are improved with a larger basis set. However, for relative redox potentials, the opposite is true. The results are insensitive to the force field (charges of the surroundings) used for the QM/MM calculations or whether the protein and solvent outside the QM system are relaxed or kept fixed at the crystal structure. With the best approach for relative potentials, mean absolute and maximum deviations of 0.17 and 0.44 V, respectively, are obtained after removing a systematic error of -0.55 V. Such an approach can be used to identify the correct oxidation states involved in a certain redox reaction.
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| 3. |
- Jafari, Sonia, et al.
(författare)
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Catalytic mechanism of human glyoxalase i studied by quantum-mechanical cluster calculations
- 2016
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1381-1177. ; 131, s. 18-30
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Tidskriftsartikel (refereegranskat)abstract
- Density functional theory has been used to study the mechanism and stereospecificity of the catalytic reaction of human glyoxalase I. We used the quantum mechanical cluster method to model the enzyme active site. Glyoxalase I accepts both enantiomers of the hemithioacetal between methylglyoxal and glutathione and converts them to the S-D enantiomer of lactoylglutathione. We have compared several previously suggested or alternative reaction mechanisms for both substrates on an equal footing. The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than in some inhibitor crystal structures, which we assign to differences in the electronic structure and the protonation states of the substrate. The symmetry of the active site model indicates that the enzyme can use the same reaction mechanism for the S and the R enantiomers of the substrate, but with exchanged roles of the two active-site glutamate residues. However, the calculations show some asymmetry (0-4 kcal mol-1 differences in reaction energies and activation barriers), caused by the different coordination states of the glutamate residues in the starting crystal structure. Our results indicate that the only possibility for the stereospecificity of glyoxalase I is differences in the electrostatic surroundings and flexibility of the glutamate residues in the active site owing to their neighboring residues in the protein.
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| 4. |
- Jafari, Sonia, et al.
(författare)
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Higher Flexibility of Glu-172 Explains the Unusual Stereospecificity of Glyoxalase i
- 2018
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; 57:9, s. 4944-4958
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Tidskriftsartikel (refereegranskat)abstract
- Despite many studies during the latest two decades, the reason for the unusual stereospecificity of glyoxalase I (GlxI) is still unknown. This metalloenzyme converts both enantiomers of its natural substrate to only one enantiomer of its product. In addition, GlxI catalyzes reactions involving some substrate and product analogues with a stereospecificity similar to that of its natural substrate reaction. For example, the enzyme exchanges the pro-S, but not the pro-R, hydroxymethyl proton of glutathiohydroxyacetone (HOC-SG) with a deuterium from D2O. To find some clues to the unusual stereospecificity of GlxI, we have studied the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by this enzyme. We employed density functional theory and molecular dynamics (MD) simulations to study the proton exchange mechanism and origin of the stereospecificity. The results show that a rigid cluster model with the same flexibility for the two active-site glutamate residues cannot explain the unusual stereospecificity of GlxI. However, using a cluster model with full flexibility of Glu-172 or a larger model with the entire glutamates, extending the backbone into the neighboring residues, the results showed that there is no way for HOC-SG to exchange its protons if the alcoholic proton is directed toward Glu-99. However, if the hydroxymethyl proton instead is directed toward the more flexible Glu-172, we find a catalytic reaction mechanism for the exchange of the HS proton by a deuterium, in accordance with experimental findings. Thus, our results indicate that the special stereospecificity of GlxI is caused by the more flexible environment of Glu-172 in comparison to that of Glu-99. This higher flexibility of Glu-172 is also confirmed by MD simulations. We propose a reaction mechanism for the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by GlxI with an overall energy barrier of 15 kcal/mol.
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| 5. |
- Jafari, Sonia, et al.
(författare)
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QM/MM study of the catalytic reaction of aphid myrosinase
- 2024
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Ingår i: International Journal of Biological Macromolecules. - 0141-8130. ; 262
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Tidskriftsartikel (refereegranskat)abstract
- Brevicoryne brassicae, an aphid species, exclusively consumes plants from the Brassicaceae family and employs a sophisticated defense mechanism involving a myrosinase enzyme that breaks down glucosinolates obtained from its host plants. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to study the catalytic reaction of aphid myrosinase. A proper QM region to study the myrosinase reaction should contain the whole substrate, models of Gln-19, His-122, Asp-124, Asn-166, Glu-167, Lys-173, Tyr-180, Val-228, Tyr-309, Tyr-346, Ile-347, Glu-374, Glu-423, Trp-424, and a water molecule. The calculations show that Asp-124 and Glu-423 must be charged, His-122 must be protonated on NE2, and Glu-167 must be protonated on OE2. Our model reproduces the anomeric retaining characteristic of myrosinase and indicates that the deglycosylation reaction is the rate-determining step of the reaction. Based on the calculations, we propose a reaction mechanism for aphid myrosinase-mediated hydrolysis of glucosinolates with an overall barrier of 15.2 kcal/mol. According to the results, removing a proton from Arg-312 or altering it to valine or methionine increases glycosylation barriers but decreases the deglycosylation barrier.
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| 6. |
- Jafari, Sonia, et al.
(författare)
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QM/MM Study of the Catalytic Reaction of Myrosinase; Importance of Assigning Proper Protonation States of Active-Site Residues
- 2021
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 17:3, s. 1822-1841
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Tidskriftsartikel (refereegranskat)abstract
- Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of β-d-S-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be protonated on the NE2 atom. The results indicate that assigning proper protonation states of the residues is more important than the size of the model QM system. Our model reproduces the anomeric retaining characteristic of myrosinase and also reproduces the experimental fact that ascorbate increases the rate of the reaction. A water molecule in the active site, positioned by Gln-187, helps the aglycon moiety of the substrate to stabilize the buildup of negative charge during the glycosylation reaction and this in turn makes the moiety a better leaving group. The water molecule also lowers the glycosylation barrier by ∼9 kcal/mol. The results indicate that the Q187E and E464A mutants but not the Q187A mutant can perform the glycosylation step. However, the energy profiles for the deglycosylation step of the mutants are not similar to that of the wild-type enzyme. The Glu-464 residue lowers the barriers of the glycosylation and deglycosylation steps. The ascorbate ion can act as a general base in the reaction of the wild-type enzyme only if the Glu-464 and His-141 residues are properly protonated.
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| 7. |
- Jafari, Sonia, et al.
(författare)
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QM/MM study of the stereospecific proton exchange of glutathiohydroxyacetone by glyoxalase I
- 2019
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Ingår i: Results in Chemistry. - : Elsevier BV. - 2211-7156. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- We have performed quantum mechanics (QM), molecular mechanics (MM) and hybrid QM/MM calculations to study the stereospecific proton exchange of glutathiohydroxyacetone (HOC-SG) by glyoxalase I (GlxI). We did the QM/MM calculations with a large QM system (246 atoms) to investigate the proton-exchange mechanism. Moreover, single-point big-QM energies with 1303 atoms in the big QM system and 22,412 atoms in the MM sys- tem were used to compare the energy difference of the stationary structures. GlxI catalyzes the exchange of the pro-S, but not the pro-R hydroxymethyl proton of HOC-SG with a deuterium from the D2O solvent. Classical mo- lecular dynamics simulations with different protonation states of Glu99, Glu172 and HOC-SG led to the determi- nation of most stable species (Glu-172 is protonated and the alcoholic oxygen of HOC-SG is deprotonated). The QM/MM results showed that before binding of HOC-SG, both active-site glutamates are charged, whereas HOC- SG is protonated. When HOC-SG binds, its alcoholic proton (HO) can point toward either Glu-99 or Glu-172. How- ever, if the substrate binds so that HO is directed toward Glu-99, it is not transferred, whereas if it is directed to- ward Glu-172, the latter abstracts HO. The results showed that transferring HO to the glutamates from the reactant states is the key step to make the proton exchange reaction possible. Our calculations show that order of basicity of the glutamates and HOC-SG inside the enzyme is: Glu-172 N HOC-SG N Glu-99. The calculations allow us to propose a reaction mechanism for the stereospecific proton exchange of HOC-SG by GlxI with an over- all barrier of 14.1 kcal/mol.
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| 8. |
- Jafari, Sonia, et al.
(författare)
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Quantum Mechanics/Molecular Mechanics Study of the Reaction Mechanism of Glyoxalase I
- 2020
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 1520-510X .- 0020-1669. ; 59, s. 2594-2603
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Tidskriftsartikel (refereegranskat)abstract
- Glyoxalase I (GlxI) is a member of the glyoxalasesystem, which is important in cell detoxification and convertshemithioacetals of methylglyoxal (a cytotoxic byproduct of sugarmetabolism that may react with DNA or proteins and introducenucleic acid strand breaks, elevated mutation frequencies, andstructural or functional changes of the proteins) and glutathioneinto D-lactate. GlxI accepts both the S and R enantiomers ofhemithioacetal, but converts them to only the S-D enantiomer oflactoylglutathione. Interestingly, the enzyme shows this unusualspecificity with a rather symmetric active site (a Zn ioncoordinated to two glutamate residues; Glu-99 and Glu-172),making the investigation of its reaction mechanism challenging.Herein, we have performed a series of combined quantummechanics and molecular mechanics calculations to study the reaction mechanism of GlxI. The substrate can bind to the enzyme in two different modes, depending on the direction of its alcoholic proton (H2; toward Glu-99 or Glu-172). Our results show that the S substrate can react only if H2 is directed toward Glu-99 and the R substrate only if H2 is directed toward Glu-172. In both cases, the reactions lead to the experimentally observed S-D enantiomer of the product. In addition, the results do not show any low- energy paths to the wrong enantiomer of the product from neither the S nor the R substrate. Previous studies have presented several opposing mechanisms for the conversion of R and S enantiomers of the substrate to the correct enantiomer of the product. Our results confirm one of them for the S substrate, but propose a new one for the R substrate.
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| 9. |
- Jafari, Sonia, et al.
(författare)
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Two local minima for structures of [4Fe–4S] clusters obtained with density functional theory methods
- 2023
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Ingår i: Scientific Reports. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- [4Fe–4S] clusters are essential cofactors in many proteins involved in biological redox-active processes. Density functional theory (DFT) methods are widely used to study these clusters. Previous investigations have indicated that there exist two local minima for these clusters in proteins. We perform a detailed study of these minima in five proteins and two oxidation states, using combined quantum mechanical and molecular mechanical (QM/MM) methods. We show that one local minimum (L state) has longer Fe–Fe distances than the other (S state), and that the L state is more stable for all cases studied. We also show that some DFT methods may only obtain the L state, while others may obtain both states. Our work provides new insights into the structural diversity and stability of [4Fe–4S] clusters in proteins, and highlights the importance of reliable DFT methods and geometry optimization. We recommend r2SCAN for optimizing [4Fe-4S] clusters in proteins, which gives the most accurate structures for the five proteins studied.
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| 10. |
- Jafari, Sonia, et al.
(författare)
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Two-Substrate Glyoxalase i Mechanism : A Quantum Mechanics/Molecular Mechanics Study
- 2021
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; , s. 303-314
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Tidskriftsartikel (refereegranskat)abstract
- Glyoxalase I (GlxI) is an important enzyme that catalyzes the detoxification of methylglyoxal (MG) with the help of glutathione (H-SG). It is currently unclear whether MG and H-SG are substrates of GlxI or whether the enzyme processes hemithioacetal (HTA), which is nonenzymatically formed from MG and H-SG. Most previous studies have concentrated on the latter mechanism. Here, we study the two-substrate reaction mechanism of GlxI from humans (HuGlxI) and corn (ZmGlxI), which are Zn(II)-active and -inactive, respectively. Hybrid quantum mechanics/molecular mechanics calculations were used to obtain geometrical structures of the stationary points along reaction paths, and big quantum mechanical systems with more than 1000 atoms and free-energy perturbations were used to improve the quality of the calculated energies. We studied, on an equal footing, all reasonable reaction paths to the S- and R-enantiomers of HTA from MG and H-SG (the latter was considered in two different binding modes). The results indicate that the MG and H-SG reaction in both enzymes can follow the same path to reach S-HTA. However, the respective overall barriers and reaction energies are different for the two enzymes (6.1 and -9.8 kcal/mol for HuGlxI and 15.7 and -2.2 kcal/mol for ZmGlxI). The first reaction step to produce S-HTA is facilitated by a crystal water molecule that forms hydrogen bonds with a Glu and a Thr residue in the active site. The two enzymes also follow similar paths to R-HTA. However, the reactions reach a deprotonated and protonated R-HTA in the human and corn enzymes, respectively. The production of deprotonated R-HTA in HuGlxI is consistent with other theoretical and experimental works. However, our calculations show a different behavior for ZmGlxI (both S- and R-HTA can be formed in the enzyme with the alcoholic proton on HTA). This implies that Glu-144 of corn GlxI is not basic enough to keep the alcoholic proton. In HuGlxI, the two binding modes of H-SG that lead to S- and R-HTA are degenerate, but the barrier leading to R-HTA is lower than the barrier to S-HTA. On the other hand, ZmGlxI prefers the binding mode, which produces S-HTA; this observation is consistent with experiments. Based on the results, we present a modification for a previously proposed two-substrate reaction mechanism for ZmGlxI.
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