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Publications (10 of 46) Show all publications
Janfalk Carlsson, Å., Bauer, P., Dobritzsch, D., Kamerlin, S. C. & Widersten, M. (2018). Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme. IUCrJ, 5(3), 269-282
Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-07-13Bibliographically approved
Hamnevik, E., Enugala, T. R., Maurer, D., Ntuku, S., Oliveira, A., Dobritzsch, D. & Widersten, M. (2017). Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer. The FEBS Journal, 284(22), 3895-3914
Open this publication in new window or tab >>Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
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2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed) Published
Abstract [en]

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.

Keywords
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318981 (URN)10.1111/febs.14279 (DOI)000415877100011 ()
Funder
Swedish Research Council, 621-2011-6055
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-03-09Bibliographically approved
Bauer, P., Janfalk Carlsson, Å., Amrein, B. A., Dobritzsch, D., Widersten, M. & Kamerlin, S. C. (2016). Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1. Organic and biomolecular chemistry, 14(24), 5639-5651
Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
Janfalk Carlsson, Å., Bauer, P., Dobritzsch, D., Nilsson, M., Kamerlin, S. C. & Widersten, M. (2016). Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis. ChemBioChem (Print), 17(18), 1693-1697
Open this publication in new window or tab >>Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
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2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-298675 (URN)10.1002/cbic.201600330 (DOI)000384425400004 ()27383542 (PubMedID)
Funder
Swedish Research CouncilEU, European Research Council, 306474Swedish National Infrastructure for Computing (SNIC), SNIC2015-16-12EU, FP7, Seventh Framework Programme, 283570
Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2017-11-28Bibliographically approved
Ma, H., Szeler, K., Kamerlin, S. C. & Widersten, M. (2016). Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA). Chemical Science, 7, 1415-1421
Open this publication in new window or tab >>Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA)
2016 (English)In: Chemical Science, ISSN 1742-2183, Vol. 7, p. 1415-1421Article in journal (Refereed) Published
Abstract [en]

DERA, 2-deoxyribose-5-phosphate aldolase, catalyzes the retro-aldol cleavage of 2-deoxy-ribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde in a branch of the pentose phosphate pathway. In addition to the physiological reaction, DERA also catalyzes the reverse addition reaction and, hence, is an interesting candidate for biocatalysis of carboligation reactions, which are central to synthetic chemistry. An obstacle to overcome for this enzyme to become a truly useful biocatalyst, however, is to relax the very strict dependency of this enzyme on phoshorylated substrates. We have studied herein the role of the non-canonical phosphate-binding site of this enzyme, consisting of Ser238 and Ser239, by site-directed and site-saturation mutagenesis, coupled to kinetic analysis of mutants. In addition, we have performed molecular dynamics simulations on the wild-type and four mutant enzymes, to analyse how mutations at this phosphate-binding site may affect the protein structure and dynamics. Further examination of the S239P mutant revealed that this variant increases the enthalpy change at the transition state, relative to the wild-type enzyme, but concomitant loss in entropy causes an overall relative loss in the TS free energy change. This entropy loss, as measured by the temperature dependence of catalysed rates, was mirrored in both a drastic loss in dynamics of the enzyme, which contributes to phosphate binding, as well as an overall loss in anti-correlated motions distributed over the entire protein. Our combined data suggests that the degree of anticorrelated motions within the DERA structure is coupled to catalytic efficiency in the DERA-catalyzed retro-aldol cleavage reaction, and can be manipulated for engineering purposes.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-266899 (URN)10.1039/C5SC03666F (DOI)000368835300072 ()
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), SNIC 2014/11-2
Available from: 2015-11-13 Created: 2015-11-13 Last updated: 2016-04-07
Ma, H., Enugala, T. R. & Widersten, M. (2015). A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates. ChemBioChem (Print), 16(18), 2595-2598
Open this publication in new window or tab >>A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates
2015 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 18, p. 2595-2598Article in journal (Refereed) Published
Abstract [en]

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose-6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been presented as a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with these type of bulky acceptor substrates is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here, we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an in vitro evolved aldehyde reductase that reduces the aldehyde product formed after FSA-afforded cleavage of the aldol. The assay is suitable both for steady state enzyme kinetics and real-time activity screening in a 96-well format.

Keywords
aldolase, dehydrogenase, directed evolution, retro-aldol, screening
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-264195 (URN)10.1002/cbic.201500466 (DOI)000367720300008 ()26449620 (PubMedID)
Funder
Swedish Research Council, 621-2011-6055
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
Karlsson, O. A., Ramirez, J., Öberg, D., Malmqvist, T., Engström, Å., Friberg, M., . . . Jemth, P. (2015). Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus. Scientific Reports, 5, Article ID 9382.
Open this publication in new window or tab >>Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 9382Article in journal (Refereed) Published
Abstract [en]

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (K-d = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-258849 (URN)10.1038/srep09382 (DOI)000351373500005 ()25797137 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2015-07-23 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
Dahlström, K. M., Blikstad, C., Widersten, M. & Salminen, T. A. (2015). Directed evolution on FucO - structural explanations for changes in substrate scope. Paper presented at 29th Annual Symposium of the Protein-Society, JUL 22-25, 2015, Barcelona, SPAIN. Protein Science, 24, 199-200
Open this publication in new window or tab >>Directed evolution on FucO - structural explanations for changes in substrate scope
2015 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, p. 199-200Article in journal, Meeting abstract (Other academic) Published
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-272104 (URN)000363658100329 ()
Conference
29th Annual Symposium of the Protein-Society, JUL 22-25, 2015, Barcelona, SPAIN
Available from: 2016-01-12 Created: 2016-01-12 Last updated: 2017-11-30Bibliographically approved
Amrein, B. A., Bauer, P., Duarte, F., Janfalk Carlsson, Å., Naworyta, A., Mowbray, S. L., . . . Kamerlin, S. C. L. (2015). Expanding the catalytic triad in epoxide hydrolases and related enzymes. ACS Catalysis, 5(10), 5702-5713
Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
Funder
EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
Hamnevik, E., Blikstad, C., Norrehed, S. & Widersten, M. (2014). Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A. Journal of Molecular Catalysis B: Enzymatic, 99, 68-78
Open this publication in new window or tab >>Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
2014 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, p. 68-78Article in journal (Refereed) Published
Abstract [en]

An increasing interest in biocatalysis and the use of stereoselective alcohol dehydrogenases in synthetic asymmetric catalysis motivates detailed studies of potentially useful enzymes such as alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber. This enzyme is capable of catalyzing enantio-, and regioselective production of phenyl-substituted α-hydroxy ketones (acyloins) which are precursors for the synthesis of a range of biologically active compounds. In this study, we have determined the enzyme activity for a selection of phenyl-substituted vicinal diols and other aryl- or alkyl-substituted alcohols and ketones. In addition, the kinetic mechanism for the oxidation of (R)- and (S)-1-phenylethanol and the reduction of acetophenone has been identified as an Iso Theorell-Chance (hit and run) mechanism with conformational changes of the enzyme-coenzyme binary complexes as rate-determining for the oxidation of (S)-1-phenylethanol and the reduction of acetophenone. The underlying cause of the 270-fold enantiopreference for the (S)-enantiomer of 1-phenylethanol has been attributed to non-productive binding of the R-enantiomer. We have also shown that it is possible to tune the direction of the redox chemistry by adjusting pH with the oxidative reaction being favored at pH values above 7.

Keywords
alcohol dehydrogenase, kinetic mechanism, pre-steady state kinetics, product inhibition
National Category
Other Chemistry Topics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-207474 (URN)10.1016/j.molcatb.2013.10.023 (DOI)000331340500010 ()
Available from: 2013-09-15 Created: 2013-09-15 Last updated: 2017-12-06Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-3203-3793

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