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  • 1.
    Amrein, Beat A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    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.

  • 2.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 12016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed)
    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.

  • 3.
    BJORNESTEDT, R
    et al.
    Uppsala University.
    STENBERG, G
    Uppsala University.
    WIDERSTEN, M
    Uppsala University.
    BOARD, PG
    Uppsala University.
    SINNING, I
    Uppsala University.
    JONES, TA
    Uppsala University.
    MANNERVIK, B
    Uppsala University.
    FUNCTIONAL-SIGNIFICANCE OF ARGININE-15 IN THE ACTIVE-SITE OF HUMAN CLASS-ALPHA GLUTATHIONE TRANSFERASE A1-11995In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 247, no 4, p. 765-773Article in journal (Other academic)
    Abstract [en]

    Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase Al-1 have shown that N-epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivati

  • 4.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Kärthe M.
    Åbo Akademi.
    Salminen, Tiina A.
    Åbo Akademi.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stereoselective oxidation of aryl-substituted vicinal diols into chiral α-hydroxy aldehydes by re-engineered propanediol oxidoreductase2013In: ACS Catalysis, ISSN 2155-5435, Vol. 3, no 12, p. 3016-3025Article in journal (Refereed)
    Abstract [en]

    α-Hydroxy aldehydes are chiral building blocks used in synthesis of natural products and synthetic drugs. One route to their production is by regioselective oxidation of vicinal diols and, in this work, we aimed to perform the oxidation of 3-phenyl-1,2-propanediol into the corresponding α‑hydroxy aldehyde applying enzyme catalysis. Propanediol oxidoreductase from E. coli efficiently catalyzes the stereoselective oxidation of S-1,2-propanediol into S-lactaldehyde. The enzyme, however, shows no detectable activity with aryl-substituted or other bulky alcohols. We conducted ISM-driven directed evolution on FucO and were able to isolate several mutants that were active with S-3-phenyl-1,2-propanediol. The most efficient variant displayed a kcat/KM of 40 s-1M-1 and the most enantioselective variant an E-value (S/R) of 80. Furthermore, other isolated variants showed up to 4400-fold increased activity with another bulky substrate, phenylacetaldehyde. The results with engineered propanediol oxidoreductases identified amino acids important for substrate selectivity and asymmetric synthesis of aryl-substituted α-hydroxy aldehydes. In conclusion, our study demonstrates the feasibility of tailoring the catalytic properties of propanediol oxidoreductase for biocatalytic properties.

  • 5.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Käthe
    Salminen, Tiina
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, p. 2387-2398Article in journal (Refereed)
    Abstract [en]

    We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

  • 6.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Functional characterization of a stereospecific diol dehydrogenase, FucO, from Escherichia coli: substrate specificity, pH dependence, kinetic isotope effects and influence of solvent viscosity2010In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 66, no 1-2, p. 148-155Article in journal (Refereed)
    Abstract [en]

    FucO, (S)-1,2-propanediol oxidoreductase, from Escherichia coli is involved in the anaerobic catabolic metabolism of L-fucose and L-rhamnose, catalyzing the interconversion of lactaldehyde to propanediol. The enzyme is specific for the S-enantiomers of the diol and aldehyde suggesting stereospecificity in catalysis. We have studied the enzyme kinetics of FucO with a spectrum of putative alcohol and aldehyde substrates to map the substrate specificity space. Additionally, for a more detailed analysis of the kinetic mechanism, pH dependence of catalysis, stereochemistry in hydride transfer, deuterium kinetic isotope effect of hydride transfer and effect of increasing solvent viscosity were also analyzed. The outcome of this study can be summarized as follows: FucO is highly stereospecific with the highest E-value measured to be 320 for the S-enantiomer of 1,2-propanediol. The enzyme is strictly regiospecific for oxidation of primary alcohols. The enzyme prefers short-chained (2-4 carbons) substrates and does not act on bulkier compounds such as phenyl-substituted alcohols. FucO is an 'A-side' dehydrogenase transferring the pro-R-hydrogen of NADH to the aldehyde substrate. The deuterium KIEs of kcat and kcat/KM were 1.9 and 4.2, respectively, illustrating that hydride transfer is partially rate-limiting but also that other reaction steps contribute to rate limitation of catalysis. Combining the KIE results with the observed effects of increasing medium viscosity proposed a working model for the kinetic mechanism involving slow, rate-limiting, product release and on-pathway conformational changes in the enzyme-nucleotide complexes.

  • 7. Cassimjee, Karim Engelmark
    et al.
    Kourist, Robert
    Lindberg, Diana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Wittrup Larsen, Marianne
    Thanh, Nguyen Hong
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Bornscheuer, Uwe T
    Berglund, Per
    One-step enzyme extraction and immobilization for biocatalysis applications2011In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 4, p. 463-469Article in journal (Refereed)
    Abstract [en]

    An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

  • 8.
    Chaga, Grigoriy
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Andersson, Lennart
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Porath, Jerker
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes1994In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 7, no 9, p. 1115-1119Article in journal (Refereed)
    Abstract [en]

    Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.

  • 9.
    Dahlström, Kathe M.
    et al.
    Abo Akad Univ, Struct Bioinformat Lab, Biochem, Turku, Finland..
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Salminen, Tiina A.
    Abo Akad Univ, Struct Bioinformat Lab, Biochem, Turku, Finland..
    Directed evolution on FucO - structural explanations for changes in substrate scope2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, p. 199-200Article in journal (Other academic)
  • 10.
    Eklund, Sandra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lindås, Ann-Christin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Tomkinson, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics2012In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1824, no 4, p. 561-570Article in journal (Refereed)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.

  • 11.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Catalysis of potato epoxide hydrolase, StEH12005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 390, p. 633-640Article in journal (Refereed)
    Abstract [en]

    The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 mmol·min-1·mg-1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

  • 12.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Widersten, Mikael
    Implications for an Ionized Alkyl-Enzyme Intermediate during StEH1-Catalyzed trans-Stilbene Oxide Hydrolysis2006In: Biochemistry, Vol. 45, p. 205-212Article in journal (Refereed)
    Abstract [en]

    The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.

  • 13.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an 'orphaned' epoxide hydrolase2005In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1748, p. 213-221Article in journal (Refereed)
    Abstract [en]

    The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterialdehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the a/hhydrolasefold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of theprotein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c proteindisplayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide.Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a newsubclass of a/h-hydrolase fold enzymes.

  • 14.
    Frickel, EM
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Jemth, P
    Widersten, M
    Mannervik, B
    Yeast glyoxalase I is a monomeric enzyme with two active sites2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, ISSN 0021-9258, Vol. 276, no 3, p. 1845-1849Article in journal (Refereed)
    Abstract [en]

    The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes, The model suggests that yeast glyoxalase I has two active sites conta

  • 15.
    Gurell, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Modification of substrate specificity resulted in an epoxide hydrolase with shifted enantiopreference for (2,3-epoxypropyl)benzene2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 10, p. 1422-1429Article in journal (Refereed)
    Abstract [en]

    Random mutagenesis targeted at hot spots of non-catalytic active-site residues of potato epoxide hydrolase StEH1 combined with an enzyme-activity screen allowed for isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the variant displaying most radical functional changes, showed a 15:1 preference for the R-enantiomer. This mutant had accumulated four substitutions distributed to two, out of four mutated, hot spots: W106L, L109Y, V141K and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S-enantiomer combined with retained activity with the R-enantiomer. The results demonstrate the feasibility to mold stereoselectivity in this biocatalytically relevant enzyme.

  • 16.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Norrehed, Sara
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A2014In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, p. 68-78Article in journal (Refereed)
    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.

  • 17.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ntuku, Siphosethu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed)
    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.

  • 18.
    Hansson, LO
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Bolton-Grob, R
    Widersten, M
    Mannervik, B
    Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene1999In: PROTEIN SCIENCE, ISSN 0961-8368, Vol. 8, no 12, p. 2742-2750Article in journal (Refereed)
    Abstract [en]

    Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosogunnidine (cyanoDMNG), and 1,2-dichloro-4-ni

  • 19.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreeen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Nilsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis2016In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed)
    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.

  • 20.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme2018In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed)
    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.

  • 21.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ma, Huan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 38, p. 7627-7637Article in journal (Refereed)
    Abstract [en]

    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.

  • 22.
    Jiang, FY
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Wisen, S
    Widersten, M
    Bergman, B
    Mannervik, B
    Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, ISSN 0022-2836, Vol. 301, no 4, p. 783-793Article in journal (Refereed)
    Abstract [en]

    A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N-8)-TACA, was found to be the optimal NtcA-binding sequence. The mor

  • 23.
    Johansson, Ann-Sofie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 1051998In: Journal of Molecular Cell Biology, ISSN 1674-2788, E-ISSN 1759-4685, Vol. 278, no 3, p. 687-698Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

  • 24.
    Jonas, Kristina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Van Der Vries, Erhard
    Nilsson, Mikael T.I.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics2005In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 18, no 11, p. 537-546Article in journal (Refereed)
    Abstract [en]

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

  • 25.
    Karlsson, O. Andreas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ramirez, Juan
    Öberg, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Malmqvist, Tony
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Friberg, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Trave, Gilles
    Nilsson, Mikael T. I.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 9382Article in journal (Refereed)
    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.

  • 26.
    Lewandowicz, Andrzej
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Rudzinski, Juliusz
    Tronstad, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ryberg, Per
    Matsson, Olle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Paneth, Piotr
    Chlorine kinetic isotope effects on the haloalkane dehalogenase reaction2001In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 123, no 19, p. 4550-4555Article in journal (Refereed)
    Abstract [en]

    We have found chlorine kinetic isotope effects on the dehalogenation catalyzed by haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 to be 1.0045 ± 0.0004 for 1,2-dichloroethane and 1.0066 ± 0.0004 for 1-chlorobutane. The latter isotope effect approaches the intrinsic chlorine kinetic isotope effect for the dehalogenation step. The intrinsic isotope effect has been modeled using semiempirical and DFT theory levels using the ONIOM QM/QM scheme. Our results indicate that the dehalogenation step is reversible; the overall irreversibility of the enzyme-catalyzed reaction is brought about by a step following the dehalogenation.

  • 27.
    Lindberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ahmad, Shabbir
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis2010In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 495, no 2, p. 165-173Article in journal (Refereed)
    Abstract [en]

    Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(1). Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the alpha/beta-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys(179)Asp(202), Glu(215)-Arg(41) and Arg(236)-Glu(136) were disrupted by mutations, K179Q E215Q, R236Q and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.

  • 28.
    Lindberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    de la Fuente Revenga, Mario
    Instituto de Quimica Medica-CSIC Juan de la Cierva 3, E-28006 Madrid, Spain.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis2010In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 147, no 3-4, p. 169-171Article in journal (Refereed)
    Abstract [en]

    A special group of ionic liquids, deep eutectic solvents (DESs) have been tested as cosolvents in enzyme-catalyzed hydrolysis of a chiral (1,2)-trans-2-methylstyrene oxide. The choline chloride:ethane diol (ET), choline chloride:glycerol (GLY) and choline:chloride:urea (REL) DESs were included in the reaction mixtures with epoxide and the potato epoxide hydrolase StEH1. The effect of the DESs on enzyme function was primarily elevations of KM (up to 20-fold) and with lesser effects on turnover numbers (two-fold variation). The regioselectivity in hydrolysis of the (1R,2R)-2-trans-methylstyrene oxide was altered in the presence of GLY or ET to favor epoxide ring opening at the benzylic carbon (R=2.33), enhancing the regioselectivity observed in buffer-only systems (R=1.35). The DES solutions dissolved 1.5-fold higher epoxide concentrations as compared to phosphate buffer. The total conversion of high concentration (40 g/l) of (1S,2S)-MeSO was not negatively affected by addition of 40 % GLY.

  • 29.
    Lindberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    de la Fuente Revenga, Mario
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Temperature and pH dependence of enzyme catalyzed hydrolysis of trans-methylstyrene oxide: a unifying kinetic model for observed hysteresis, cooperativity and regioselectivity2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 10, p. 2297-2304Article in journal (Refereed)
    Abstract [en]

    The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide hydrolases towards certain epoxides has been studied. The effects of temperature and pH on regioselectivity was investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14 to 46 °C and pH 6.0 to 9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures ≤30 °C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will be dependent on the relationship between kcat and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways towards product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also display distinct pH dependencies of kcat/KM. The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that 1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both ΔΔH and ΔΔS, and 2) the hysteretic behavior is linked to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.

  • 30.
    Lindberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 24, p. 6309-6320Article in journal (Refereed)
    Abstract [en]

    The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by 13C-NMR spectroscopy of 18O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency.

  • 31.
    Ma, Huan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates2015In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 18, p. 2595-2598Article in journal (Refereed)
    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.

  • 32.
    Ma, Huan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Szeler, Klaudia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA)2016In: Chemical Science, ISSN 1742-2183, Vol. 7, p. 1415-1421Article in journal (Refereed)
    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.

  • 33.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bernhard, Lohkamp
    Krumpel, Michael
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Characterization and structure determination of human β-ureidopropionase reveal pH-dependent regulation by ligand-induced changes in oligomerizationManuscript (preprint) (Other academic)
  • 34.
    Mowbray, Sherry L.
    et al.
    Swedish University of Agricultural Sciences, Department of Molecular Biology.
    Elfström, Lisa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ahlgren, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andersson, Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    X-ray structure of potato epoxide hydrolase sheds light on its substrate specificity2006In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, no 7, p. 1628-1637Article in journal (Refereed)
    Abstract [en]

    Abstract: Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refined to a resolution of 1.95 angstrom. Analysis of the structure allows a better understanding of the observed substrate specificities and activity. Further, comparisons with mammalian and fungal epoxide hydrolase structures reported earlier show the basis of differing substrate specificities in the various epoxide hydrolase subfamilies. Most plant enzymes, like the potato epoxide hydrolase, are expected to be monomers with a preference for substrates with long lipid-like substituents of the epoxide ring. The significance of these results in the context of biological roles and industrial applications is discussed.

  • 35. Mukanganyama, Stanley
    et al.
    Widersten, Mikael
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Naik, Yogeshkumar S.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Hasler, Julia A.
    Inhibition of glutathione S-transferases by antimalarial drugs: Possible implications for circumventing anticancer drug resistance2002In: Int J Cancer, Vol. 97, p. 700-705Article in journal (Refereed)
  • 36.
    Nilsson, Mikael T.I.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mossing, Michael C.
    Widersten, Mikael
    Institutionen för naturvetenskaplig biokemi, Department of Biochemistry.
    Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins2000In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, ISSN 0269-2139, Vol. 13, no 7, p. 519-526Article in journal (Refereed)
    Abstract [en]

    A robust selection system affording phage display of the DNA-binding helix–turn–helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.

  • 37.
    Nilsson, Mikael T.I.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Repertoire selection of variant single-chain Cro: towards directed DNA-binding specificity of helix-turn-helix proteins2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 38, p. 12038-12047Article in journal (Refereed)
    Abstract [en]

    A single-chain derivative of the lambda Cro repressor (scCro) has been randomly mutated in amino acid residues critical for specific DNA recognition to create libraries of protein variants. Utilizing phage display-afforded affinity selection, scCro variants have been isolated for binding to synthetic DNA ligands. Isolated scCro variants were analyzed functionally, both in fusion with phage particles and after expression of the corresponding free proteins. The binding properties with regard to specificity and affinity in binding to different DNA ligands were investigated by inhibition studies and determination of equilibrium dissociation constants for formed complexes. Variant proteins with altered DNA-sequence specificity were identified, which favored binding of targeted synthetic DNA sequences over a consensus operator sequence, bound with high affinity by wild-type Cro. The specificities were relatively modest (2-3-fold, as calculated from KD values), which can be attributed to the inherent properties in the design of the selection system; one half-site of the synthetic DNA sequences maintains the consensus operator sequence, and one "subunit" of the variant single-chain Cro dimers was conserved as wild-type sequence. The anticipated interaction between the wild-type subunit and the consensus DNA half-site of target DNA ligands is, hence, expected to contribute to the overlap in sequence discrimination. The binding affinity for the synthetic DNA sequences, however, was improved 10-30-fold in selected variant proteins as compared to "wild-type" scCro.

  • 38.
    Segura-Aguilar, Juan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Baez, Sofia
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Welch, Christopher J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Human class Mu glutathione transferases, in particular isoenzyme M2-2,catalyze detoxication of the dopamine metabolite aminochrome1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 9, p. 5727-5731Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferases (GSTs) were shown to catalyze the reductive glutathione conjugation of aminochrome (2, 3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed the highest specific activity (148 micromol/min/mg), whereas GSTs A1-1, A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1 micromol/min/mg). The product of the conjugation, with a UV spectrum exhibiting absorption peaks at 277 and 295 nm, was 4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR spectroscopy. In contrast to reduced forms of aminochrome (leucoaminochrome and o-semiquinone), 4-S-glutathionyl-5, 6-dihydroxyindoline was stable in the presence of molecular oxygen, superoxide radicals, and hydrogen peroxide. However, the strongly oxidizing complex of Mn3+ and pyrophosphate oxidizes 4-S-glutathionyl-5,6-dihydroxyindoline to 4-S-glutathionylaminochrome, a new quinone derivative with an absorption peak at 620 nm. GST M2-2 (and to a lower degree, GST M1-1) prevents the formation of reactive oxygen species linked to one-electron reduction of aminochrome catalyzed by NADPH-cytochrome P450 reductase. The results suggest that the reductive conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2, is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive oxygen species.

  • 39. Sundberg, Kathrin
    et al.
    Johansson, Ann-Sofie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Seidel, Albrecht
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Jernström, Bengt
    Differences in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons1998In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 19, no 3, p. 433-436Article in journal (Refereed)
    Abstract [en]

    Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (kcat/Km) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti- and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest kcat/Km value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionylation. In conclusion, the present study indicates that individuals who are homozygous for the allele GSTP1* B (coding for GSTP1-1/V-105) display a higher susceptibility to malignancy because of other factors than a decreased catalytic efficiency of GSTP1-1/V-105 in the detoxication of carcinogenic diol epoxides of benzo[a]pyrene or structurally related PAH.

  • 40.
    Thomaeus, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Active site of epoxide hydrolases revisited: A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 9, p. 2466-2479Article in journal (Refereed)
    Abstract [en]

    The carboxylate of Glu(35) in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of k(cat), and the rate of alkylenzyme hydrolysis, k(3). The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pK(a) of His(300) in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu(35) participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His(300).

  • 41.
    Thomaeus, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Naworyta, Agata
    Mowbray, Sherry L.
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability2008In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, no 7, p. 1275-1284Article in journal (Refereed)
    Abstract [en]

    A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro186, Leu266, His269, and the His153 imidazole. The hydroxyl of Tyr149 is also an integrated component of the chain, which leads to the hydroxyl of Tyr154. Available data suggest that Tyr154 functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 Å resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k cat/K M was similar or slightly increased compared with the wild-type reactions. k cat for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K M. Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k cat but lowered thermostability.

  • 42. Tsoi, Carrie
    et al.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Morgenstern, Ralf
    Swedmark, Stellan
    Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity.2004In: Biochemical Journal, Vol. 378, p. 687–692-Article in journal (Refereed)
    Abstract [en]

    The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure–activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in Km value) but 54-fold less efficient in sulphating dopamine (8-fold increase in Km value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.

  • 43.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Heterologous Expression in Escherichia coli of SolubleActive-Site Random Mutants of Haloalkane Dehalogenase from Xanthobacter autotrophicus GJ10 by Coexpression of Molecular Chaperonins GroEL/ES1998In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 13, no 3, p. 389-395Article in journal (Refereed)
    Abstract [en]

    A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase DhlA from Xanthobacter autotrophicus strain GJ10 is presented. Thestrategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble DhlA. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type DhlA as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)5-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant DhlA. The purified His-tagged enzyme, DhlA-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.

  • 44.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Protein engineering for development of new hydrolytic biocatalysts2014In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 21, p. 42-47Article, review/survey (Refereed)
    Abstract [en]

    Hydrolytic enzymes play important roles as biocatalysts in chemical synthesis. The chemical versatility and structurally sturdy features of Candida antarctica lipase B has placed this enzyme as a common utensil in the synthetic tool-box. In addition to catalyzing acyl transfer reactions, a number of promiscuous activities have been described recently. Some of these new enzyme activities have been amplified by mutagenesis. Epoxide hydrolases are of interest due to their potential as catalysts in asymmetric synthesis. This current update discusses recent development in the engineering of lipases and epoxide hydrolases aiming to generate new biocatalysts with refined features as compared to the wild-type enzymes. Reported progress in improvements in reaction atom economy from dynamic kinetic resolution or enantioconvergence are also included.

  • 45.
    Widersten, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gurell, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lindberg, Diana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1800, no 3, p. 316-326Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols. SCOPE OF REVIEW: This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure-activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented. MAJOR CONCLUSIONS: The present understanding of structure-activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts. GENERAL SIGNIFICANCE: The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.

  • 46. Yilmaz, Selma
    et al.
    Widersten, Mikael
    Emahazion, Tesfai
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Mannervik, Bengt
    Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase A1-11995In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 8, no 11, p. 1163-1169Article in journal (Refereed)
    Abstract [en]

    Two mutant forms of human glutathione transferase (GST) A1-1 with affinity for metal ions were constructed by introduction of His residues by site-directed mutagenesis. A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mutations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His. The mutant proteins were obtained in good yields (40-150 mg per 3 l culture) by heterologous expression in Escherichia coli. The mutant enzymes possessed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrated by immobilized metal ion affinity chromatography. The mutant with two novel His residues (2-His mutant) did not bind as tightly to immobilized Ni(II) as did the mutant with five novel His residues (5-His mutant). When tested for affinity to immobilized Zn(II), only the 5-His mutant remained bound to the column. The affinity of the 5-His mutant for Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system. Analysis of the binding curve showed two binding sites per enzyme subunit and a dissociation constant of 6.7 +/- 1.6 mu M. The kinetic constants kcat, Km and kcat/Km for the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were determined by steady-state kinetic analysis and the parameter values for the mutant forms were found to be indistinguishable from those obtained for the wild-type GST A1-1. The differences in surface charge in the mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of kcat. The results provide an alternative method for purification of fully active recombinant GST A1-1 by the introduction of novel metal binding sites. The data also showed that two His residues are sufficient for Ni(II) binding.

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