| 1. |
- Amrein, Beat A., et al.
(författare)
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Expanding the catalytic triad in epoxide hydrolases and related enzymes
- 2015
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435 .- 2155-5435. ; 5:10, s. 5702-5713
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Tidskriftsartikel (refereegranskat)abstract
- Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.
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| 2. |
- 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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| 3. |
- Ericsson, Olle, et al.
(författare)
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Clinical validation of a novel automated cell-free DNA screening assay for trisomies 21, 13, and 18 in maternal plasma.
- 2019
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Ingår i: Prenatal diagnosis. - : Wiley. - 1097-0223 .- 0197-3851. ; 39:11, s. 1011-1015
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Tidskriftsartikel (refereegranskat)abstract
- To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex.Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates.The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result.The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.
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| 4. |
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| 5. |
- Janfalk Carlsson, Åsa, 1980-, et al.
(författare)
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Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
- 2018
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Ingår i: IUCrJ. - 2052-2525. ; 5:3, s. 269-282
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Tidskriftsartikel (refereegranskat)abstract
- The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenylpropane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.
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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. |
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| 9. |
- Janfalk Carlsson, Åsa, et al.
(författare)
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Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis : contributions from changes in both enantio- and regioselectivity
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:38, s. 7627-7637
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Tidskriftsartikel (refereegranskat)abstract
- Enzyme variants of the plant epoxide hydrolase StEH1 displaying improved stereoselectivities in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene were generated by directed evolution. The evolution was driven by iterative saturation mutagenesis in combination with enzyme activity screenings where product chirality was the decisive selection criterion. Analysis of the underlying causes of the increased diol product ratios revealed two major contributing factors: increased enantioselectivity for the corresponding epoxide enantiomer(s) and, in some cases, a concomitant change in regioselectivity in the catalyzed epoxide ring-opening half-reaction. Thus, variant enzymes that catalyzed the hydrolysis of racemic (2,3-epoxypropyl)benzene into the R-diol product in an enantioconvergent manner were isolated.
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| 10. |
- Janfalk Carlsson, Åsa, 1980-
(författare)
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Towards Understanding of Selectivity & Enantioconvergence of an Epoxide Hydrolase
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1).A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for. The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5. Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104.Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering.For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.
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