| 1. |
- Zhang, Wei
(författare)
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Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles : A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.
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| 2. |
- Zuo, Shusheng, 1968-
(författare)
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Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.
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| 3. |
- Eklund, Birgitta I., 1966-
(författare)
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Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs : Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.
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| 4. |
- Fedulova, Natalia, 1980-
(författare)
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Alpha-class Glutathione Transferases from Pig: a Comparative Study
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine. A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II). In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I). Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III).
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| 5. |
- Gurell, Ann, 1981-
(författare)
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Biochemical Studies on a Plant Epoxide Hydrolase : Discovery of a Proton Entry and Exit Pathway and the Use of In vitro Evolution to Shift Enantioselectivity
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.
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| 6. |
- Hegazy, Usama M., 1973-
(författare)
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Structure-Function Relationships of Pi Class Glutathione Transferase Studied by Protein Engineering
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites.Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.
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| 7. |
- Ivarsson, Ylva, 1976-
(författare)
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Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign : A Study on Mu Class Glutathione Transferases
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.
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| 8. |
- Kurtovic, Sanela, 1979-
(författare)
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Directed Evolution of Glutathione Transferases Guided by Multivariate Data Analysis
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. Several multivariate analysis tools, e.g. principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.
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| 9. |
- Larsson, Anna-Karin, 1971-
(författare)
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Directed Enzyme Evolution of Theta Class Glutathione Transferase : Studies of Recombinant Libraries and Enhancement of Activity toward the Anticancer Drug 1,3-bis(2-Chloroethyl)-1-nitrosourea
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) are detoxication enzymes involved in the cellular protection against a wide range of reactive substances. The role of GSTs is to catalyze the conjugation of glutathione with electrophilic compounds, which generally results in less toxic products. The ability to catalyze the denitrosation of the anticancer drug 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU) was measured in twelve different GSTs. Only three of the enzymes showed any measurable activity with BCNU, of which human GST T1-1 was the most efficient. This is of special interest, since human GST T1-1 is a polymorphic protein and its expression in different patients may be crucial for the response to BCNU.DNA shuffling was used to create a mutant library by recombination of cDNA coding for two different Theta-class GSTs. In total, 94 randomly picked mutants were characterized with respect to their catalytic activity with six different substrates, expression level and sequence. A clone with only one point mutation compared to wild-type rat GST T2-2 had a significantly different substrate-activity pattern. A high expressing mutant of human GST T1-1 was also identified, which is important, since the yield of the wild-type GST T1-1 is generally low. Characterization of the Theta library demonstrated divergence of GST variants both in structure and function. The properties of every mutant were treated as a point in a six-dimensional substrate-activity space. Groups of mutants were formed based on euclidian distances and K-means cluster analyses. Both methods resulted in a set of five mutants with high alkyltransferase activities toward dichloromethane and 4-nitrophenethyl bromide (NPB). The five selected mutants were used as parental genes in a new DNA shuffling. Addition of cDNA coding for mouse and rat GST T1-1 improved the genetic diversity of the library. The evolution of GST variants was directed towards increased alkyltransferase activity including activity with the anticancer drug BCNU. NPB was used as a surrogate substrate in order to facilitate the screening process. A mutant from the second generation displayed a 65-fold increased catalytic activity with NPB as substrate compared to wild-type human GST T1-1. The BCNU activity with the same mutant had increased 175-fold, suggesting that NPB is a suitable model substrate for the anticancer drug. Further evolution presented a mutant in the fifth generation of the library with 110 times higher NPB activity than wild-type human GST T1-1.
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| 10. |
- Lindström, Helena, 1961-
(författare)
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Exploring the steroidogenic activity of glutathione transferases across species
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication. However, some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in tissues of Homo sapiens (humans), Sus scrofa (pigs) and ruminants is involved in biosynthesis of steroid hormones, catalyzing a double-bond isomerization reaction as the last step of synthesis of Δ4-pregnene-3,20-dione (progesterone) and the obligatory step in the synthesis of the last precursor of testosterone, Δ4-androstenene-3,17-dione. Steroids regulate several vital aspects of life such as for example glucose homeostasis, inflammation, immunosuppression, blood pressure, reproduction and pregnancy.The human GST A3-3 was the most efficient steroid double-bond isomerase known so far in mammals. Our work extends discoveries of GSTs that act in the steroidogenic pathways in large mammals to Equus ferus caballus (horse). The kinetic profile of EcaGST A3-3 reveals a catalytic efficiency higher than that of the human enzyme making EcaGST A3-3 the most efficient steroid double-bond isomerase known today in mammals.In contrast to the rodents, Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes it a more suitable model for human steroidogenesis than the murine one. Inhibition of EcaGST A3-3 might help treat endocrine disorders. We screened a library of 1040 FDA-approved compounds for novel inhibitors of EcaGST A3-3 and made a further characterization of the most potent inhibitors.To extend the search for steroidogenic GSTs to other mammals, we probed the degree of GST A3-3 amino acid sequence conservation in Homo sapiens, Equus ferus caballus, Canis lupus familiaris (dog), Capra hircus (goat) and Monodelphis domestica (gray short-tailed opossum). We generated expression vectors containing homologous DNA from these species to facilitate further evaluation of the activity of these GSTs in mammals.We continued to expand the research to insects by investigating the steroidogenic activity of GSTE14 in Drosophila melanogaster (fruit fly), where this enzyme has been shown to be implicated in molting.Our work has provided insights into the role of GSTs in steroidogenesis in mammals and insects, further accentuating the functional versatility of GSTs. We have provided an initial step for the development of potential treatments of steroidogenic disorders as well as tools for further investigation of activity of these GSTs in mammals.
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