Student Number : 0213014A - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers comprising two GST subunits. Interactions across the subunit interface play an important role in stabilising the subunit tertiary structure and maintain the dimeric structure required for activity. The crystal structure of a rat Mu class GST consisting of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge cluster at the subunit interface. Previous investigations revealed the lock-andkey motif was not essential for dimerisation. It was therefore postulated that the mixed-charge cluster at the dimer interface is primarily responsible for subunit association. Statistical analyses of individual rGST M1-1 chains did not predict the presence of any charge clusters. This suggests that the mixed-charge cluster forms only upon dimerisation and reinforces the probability that quaternary structure stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a structurally conserved residue in the GST family involved in the mixed-charge cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the lock-and-key motif, was mutated to serine. These changes were engineered to disrupt the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified that these engineered amino acids resulted in a stable monomeric protein (F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic activity, suggesting perturbations of the active site or substrate binding sites. Structural investigations of the monomer by far- and near-UV circular dichroism revealed a similar secondary structural content to the wild-type. However, the tryptophan fluorescence properties suggested the tryptophans were situated in more hydrophilic environments than in the wild-type. ANS binding studies indicated a large increase in the accessible hydrophobic surface area of the monomer. Ureainduced equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate unfolding model. The unfolding data indicates decreased conformational stability and a large increase in the solvent exposed surface area of the monomer. In conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the dimer and the stabilities of individual rGST M1-1 subunits.

ABSTRACT Equilibrium unfolding, single- and double-jump kinetic studies were conducted to determine the unfolding and refolding pathway of glutaredoxin 2. Structural changes for wild-type glutaredoxin 2 were monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence for equilibrium unfolding and intrinsic tryptophan fluorescence for single- and double-jump kinetics studies. Glutaredoxin 2 possesses two tryptophan residues in domain 2. In order to monitor changes in domain 1, cysteine 9 at the active site cysteines, situated in domain 1, was labelled with an extrinsic fluorophore, AEDANS, and a mutant was created (Y58W glutaredoxin 2). The AEDANS labelled protein displayed decreased alpha-helical secondary structure and conformational stability. A high degree of cooperativity and similar conformational stability was observed during the two-state transition of the urea-induced equilibrium unfolding of both the wild-type and Y58W glutaredoxin 2 proteins therefore Y58W glutaredoxin 2 could be used to assess structural changes in the local environment of domain 1 during unfolding and refolding. Two phases of unfolding, the fast and slow phase, occurred for both the wild-type and Y58W proteins. The slow phase involves structural rearrangements that expose small amounts of surface area while the fast phase represents gross structural unfolding exposing large amounts of surface area. The isomerization of the Val48-Pro49 peptide bond to the trans conformation occurs during the slow phase and this isomerization is coupled to conformational unfolding of the protein. The structural separation of these phases could be represented by two structural units (unit x and unit y), these units do not represent domain 1 and 2. The units could also result in parallel refolding pathways with the folding of the x unit involving the fast and slow refolding phases and the folding of the y unit of structure is represented by the medium phase of refolding. The fast and slow phases are further separated as the fast phase represents the gross structural folding of glutaredoxin 2 for species with the Val48-Pro49 peptide bond in the native cis conformation. The development of the slow phase after extended unfolding delay periods during double-jump refolding studies, as well as the acceleration of the rate of the phase by the peptidyl prolyl isomerase hFKBP-12 proved that the phase involves a proline peptide bond iv isomerization. This phase represents a slow isomerization coupled with conformational folding similar to the slow unfolding phase. Complex unfolding and refolding kinetics indicated the involvement of kinetic intermediates during (un)folding.

La résistance bactérienne aux antibiotiques est un problème majeur de santé publique. Un moyen de la combattre est de viser des cibles non encore exploitées pour retarder l’apparition de la résistance. Dans ce contexte, nous avons entrepris la caractérisation sur les plans biochimique et structural de l’enzyme MraY, une protéine intégrale de membrane, membre d’une famille de transférases membranaires. MraY catalyse la première étape membranaire de la biosynthèse du peptidoglycane bactérien à savoir, le transfert du motif N-acétylmuramoyl-pentapeptide du précurseur cytoplasmique UDP-MurNAc-pentapeptide sur le transporteur membranaire, l’undécaprényl-phosphate aboutissant à la formation du lipide I. Aucune structure 3D de cette enzyme n’est disponible actuellement et aucun antibiotique en utilisation clinique ne la cible. D’une part nous avons entrepris la caractérisation structurale de cette enzyme par des approches de biophysique. Des essais de cristallisation 2D dans des systèmes membranaires ont permis d’observer au microscope électronique des dimères de MraY (taille de 70Å/50Å). Des expériences de diffusion des rayons X (SAXS) montrent un rayon de giration d’environ 42Å. Les résultats issus des expériences de SAXS ont été combinés à des approches de modélisation afin déterminer l’état d’oligomérisation de cette protéine en présence de détergents. Enfin, en vue de faciliter la cristallogenèse 3D, des chimères de MraY en fusion avec des protéines hydrosolubles de structure 3D résolues (mCherry et GFP) ont été construites. Des essais de cristallisation de la protéine seule et des chimères construites ont été effectués.D’autre part, nous avons élucidé le mécanisme catalytique de l’enzyme MraY et de son paralogue WecA. Au cours de ma thèse, j’ai pu montrer que cette famille de transférase membranaire présente un mécanisme catalytique commun qui procède en une seule étape par attaque directe d’un oxyanion du substrat lipidique, préalablement déprotoné par un résidu aspartate invariant, sur le phophate Beta du substrat nucléotidique. Cela conduit à la formation du produit lipidique et libération de l’UMP. / The growing emergence of multiresistance of pathogenic bacteria to currently used antibiotics is a major public health problem that requires the development of new therapeutic compounds and the identification and exploitation of novel targets. In this context, we undertook the biochemical and structural characterization of MraY enzyme, an integral membrane protein, member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily. The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate, yielding C55-PP-MurNAc-pentapeptide (lipid I). To date, no crystal structure has been reported for this enzyme. On the one hand we have undertaken the structural characterization of this enzyme by differents biophysical approaches. Electron microscopic images after two-dimensional crystallization of the protein displayed a dimeric organisation of the MraY enzyme (size 70Å/50Å). Small X-ray scattering (SAXS) experiments have shown a radius of gyration of about 42A. The results of SAXS experiments were combined with modeling approaches to determine the oligomerization state of the protein in the presence of detergents. Finally, in order to facilitate 3D crystallisation of MraY, fusion proteins of MraY and mCherry/GFP were constructed. Crystallization trials of MraY alone and the constructed chimeras were made. On the other hand, we have elucidated the catalytic mechanism of the MraY transferase and its paralog WecA. In this study, we have shown that this family of membrane transferases has a common catalytic mechanism that proceeds by a single step displacement. During this “one-step” mechanism, the oxyanion of the poly-prenyl phosphate attacks the β phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP.

<p>A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in <i>E. coli</i>. 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.</p>

Free radicals which have critical roles in living systems through their beneficial and detrimental effects play an important role in medical revolution in health. Radicals are produced in the cells and tissues of our bodies by various processes and reactions. Diabetes mellitus is an extremely common disease in the world which seems to be accompanied by a shortage of antioxidants and an increase in free radicals, the end result of oxidative stress. Glutathione S-Transferases (GST / EC 2.5.1.18) are found in enzymatic defense system which has a role in defending cells against potentially toxic and/or carcinogenic compounds. In this study, the changes in the activities and expressions of various GST isozymes in the liver of diabetic rats related to oxidative stress were studied. The effects of antioxidants, Vitamin C and &amp / #945 / -Lipoic acid on GST isozyme activities and mRNA expressions were also investigated. According to our results, diabetic rats exhibited decreased mRNA expressions of both GSTA2 and GSTM1 genes, but the activities of only GST Mu isozyme decreased in diabetic rats, compared to controls and GST Alpha isozyme activity remained unchanged in diabetic animals. Our results also showed that &amp / #945 / -Lipoic acid individually has no significant effect on both GSTA2 and GSTM1 gene expressions and activities. Furthermore, although the administration of Vitamin C alone showed no significant effect on all GST isozyme activities, it decreased GSTA2 mRNA expression significantly. The administration of Vitamin C and &amp / #945 / -Lipoic acid together affected both GSTA2 and GSTM1 mRNA expressions in control rats, but only GST Mu activity showed a significant change. The results of this study showed that, the administration of two antioxidants, &amp / #945 / -Lipoic acid and Vitamin C alone and together did not reverse the results of diabetes at the level of both gene expression and activities of GST isozymes.

Protein farnesylation is an essential post-translational modification required for the function of numerous cellular proteins including the oncoprotein Ras. The farnesyl transferase (FTase) catalyzed reaction is unique because farnesyl diphosphate (FPP), the farnesyl group donor for the reaction, forms a significant portion of a target protein binding site. The major goal of this research was to exploit this unique property of the FTase reaction and determine if changing the structure of the farnesyl donor group would affect FTase protein targeting. A small library of structural analogues of FPP was synthesized. Michelis-Menten steady-state kinetic analyses and competition reactions were used to determine the effect of these structural modifications on FTase targeting. We found that the analogues did affect FTase protein selectivity and that this could be exploited to induce unnatural target selectivity into the enzyme. The second goal of this research was to determine the effect of FPP analogues on the function of FTase target proteins. To test the effect of these analogues we determined whether the unnatural lipid could ablate oncogenic H-Ras biological function in a Xenopus laevis model system. Several analogues were able to disrupt oncogenic H-Ras function while others mimicked the activity of FPP. These results indicated that some of the FPP analogues may act a prenyl group function inhibitors that could lead to an important new class of anti-cancer therapeutics. Another major goal of this research was to use the FPP analogues as unnatural probes for the endogenous cellular activity of FTase target proteins. We developed antibodies to two of the unnatural FPP analogues to study their activity in cell cultureUtilizing these antibodies we found that alcohol prodrugs of the FPP analogues could be incorporated into cellular proteins in an FTase dependent manner. The ability of cell permeant analogues to be incorporated into live cells enhances the chances that such a molecule could be used to modify oncogenic cellular proteins with a prenyl group function inhibitor.

Plants can not escape from biotic and abiotic stress factors such as, extreme temperatures, high light intensity, drought, UV radiation, heavy metals, and pathogen attack. Plants have versatile defens systems against such stress conditions. In this study, the role of glutathione S-transferases (GSTs) in cold stress conditions were examined. Glutathione S-transferases are the enzymes that detoxify natural and exogenous toxic compounds by conjugation with glutathione. Glutathione, an endogenous tripeptide, is important as reducing agent, nucleophilic scavenger, and alleviate the chemical toxicity in the plants by the reaction of GSTs. Glutathione conjugates can be transported to the vacuoles or apoplast and are generally much less toxic than the parent compounds. In plants there are four distinct families of the soluble GSTs, namely Phi (F), Type I / Zeta (Z), Type II / Tau (U), Type III / Theta (T), Type IV. By contrast with the mammalian families of GST, relatively little is known about the plant GST families. Up to date, there is not any study on GST isolation and characterization from Turkish red pine, in this respect, this study well play a frontier role the future research dealing with this topic. In this study, some properties of Turkish red pine GST activity towards CDNB (1-chloro-2,4 dinitrobenzene) were examined. The average specific activity of Turkish red pine GST towards CDNB was found as 200&plusmn / 50 (Mean&plusmn / SE, n= 18) nmole/min/mg cytosolic protein. GSTs in cytosol prepared from Turkish red pine needles retained its activity without loss for four weeks at -80&amp / #61616 / C. The rate of conjugation reactions were linear up to 0.8mg of Turkish red pine cytosolic protein and 0.4 mg cytosolic protein was routinely used. The Turkish red pine GST showed its maximum activity at pH 8.0 in 25 mM phosphate buffer and 42 &amp / #730 / C. The measurements were carried out at room temperature (RT) of 25 &amp / #61616 / C. Turkish red pine GST seemed to be saturated at 1 mM CDNB and 1 mM GSH concentrations. The Vmax and Km values of Turkish red pine GST for CDNB was 416nmole/min/mg protein and 0,8 mM, respectively, and for GSH 106.4 nmole/min/mg protein and 0.10 mM, respectively. Turkish red pine cytosol was applied on DEAE-Sepharose fast flow column but almost no purification was achieved with respect GST activity. In order to examine the effects of cold stress on Turkish red pine GST activity, the GST activity was determined in 240 seedlings at &ndash / 3&amp / #61616 / , 0&amp / #61616 / and 13 &amp / #61616 / C environmental temperatures. It was observed that GST activity was the highest at -3&amp / #730 / C and the lowest at 13&amp / #730 / C in both cold resistant and sensitive families with the exception of Yaylaalan and &Ccedil / ameli.

Helicoverpa armigera is a polyphagous pest. Due to excessive use of insecticides, the field populations of H. armigera have become resistant to synthetic pyrethroids by one or combination of three mechanisms / reduced penetration through the cuticle, decreased nerve sensitivity and enhanced metabolism by the detoxification enzymes especially glutathione S-transferases. In this study, gut sections of H. armigera were obtained from Adana and Antalya field populations and susceptible populations from Israel. Each gut section was homogenized separately in 1.0 ml, 40 mM and pH 7.5 phosphate buffers. GST activity was determined using CDNB as substrate. Product formation linearly increased up to 29.5&micro / g proteins in 20mM, pH 7.5 phosphate buffers. Maximum reaction rate was reached at 30&amp / #9702 / C. The Vmax and Km values for GST towards CDNB and GSH were calculated with Lineweaver-Burk and Eadie-Scatchard plots as CDNB Vmax / 6.54&micro / mol/min/mg, 6.35&micro / mol/min/mg , Km / 0.29mM, 0.28mM ,respectively and as GSH Vmax / 6.42&micro / mol/min/mg, 6.65&micro / mol/min/mg, Km / 0.22mM, 0.23mM, respectively. Cytosolic GST activity of each individual from Adana, Antalya and susceptible populations were determined under optimized conditions. The mean of GST activity in Adana population (n=50) and Antalya population (n=50) were found 7.824&micro / mol/min/mg and 9.518&micro / mol/min/mg, respectively. The mean of GST activity in susceptible population (n=50) was determined as 3.272&micro / mol/min/mg. According to these results, GST activities of Adana and Antalya field populations&rsquo / showed statistically significant increase (p&lt / 0.05) than susceptible H. armigera populations with ANOVA method. In addition, Antalya population showed statistically increase (p&lt / 0.05) GST activity than Adana.

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.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.