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1
Shrimpton, Paul James. "A computational investigation of dihydrofolate reductase." Thesis, University of Birmingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403014.
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Haddow, J. "Potential suicide inhibitors of dihydrofolate reductase." Thesis, University of Strathclyde, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381497.
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Fan, Yongjia. "Bistability in Human Dihydrofolate Reductase Catalysis." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1280243471.
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Chui, Wai K. "Non-classical inhibitors of dihydrofolate reductase." Thesis, Aston University, 1990. http://publications.aston.ac.uk/12623/.
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This thesis comprises two main objectives. The first objective involved the stereochemical studies of chiral 4,6-diamino-1-aryl-1,2-dihydro-s-triazines and an investigation on how the different conformations of these stereoisomers may affect their binding affinity to the enzyme dihydrofolate reductase (DHFR). The ortho-substituted 1-aryl-1,2-dihydro-s-triazines were synthesised by the three component method. An ortho-substitution at the C6' position was observed when meta-azidocycloguanil was decomposed in acid. The ortho-substituent restricts free rotation and this gives rise to atropisomerism. Ortho-substituted 4,6-diamino-1-aryl-2-ethyl-1,2-dihydro-2-methyl-s-triazine contains two elements of chirality and therefore exists as four stereoisomers: (S,aR), (R,aS), (R,aR) and (S,aS). The energy barriers to rotation of these compounds were calculated by a semi-empirical molecular orbital program called MOPAC and they were found to be in excess of 23 kcal/mol. The diastereoisomers were resolved and enriched by C18 reversed phase h.p.l.c. Nuclear overhauser effect experiments revealed that (S,aR) and (R,aS) were the more stable pair of stereoisomers and therefore existed as the major component. The minor diastereoisomers showed greater binding affinity for the rat liver DHFR in in vitro assay. The second objective entailed the investigation into the possibility of retaining DHFR inhibitory activity by replacing the classical diamino heterocyclic moiety with an amidinyl group. 4-Benzylamino-3-nitro-N,N-dimethyl-phenylamidine was synthesised in two steps. One of the two phenylamidines indicated weak inhibition against the rat liver DHFR. This weak activity may be due to the failure of the inhibitor molecule to form strong hydrogen bonds with residue Glu-30 at the active site of the enzyme.
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El-Hamamsy, Mervat Hamed Rabu Ibrahem. "Potential antimycobacterial agents targeting dihydrofolate reductase." Thesis, University of Bath, 2005. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418600.
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Guo, Jian Nan. "Thermophilicity and catalytic efficiency in dihydrofolate reductase." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/56290/.
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This thesis presents an investigation of the hydrogen transfer reactions between dihydrofolate (H2F) and NADPH that are catalysed by the dihydrofolate reductase (DHFR) isolated from Geobacillus stearothermophilus (BsDHFR) as well as an artificial hybrid originating from the DHFRs from mesophilic Escherichia coli (EcDHFR) and hyperthermophilic Thermotoga maritima (TmDHFR). A broad spectrum of studies, focusing on the relationship between structure, thermostability and kinetics, showed that the catalytic behaviour of BsDHFR is generally similar to other monomeric DHFRs, including ones found in the mesophile Escherichia coli and the psychrophile Moritella profunda, but significantly different from the dimeric TmDHFR. The fact that all monomeric DHFRs display similar catalytic behaviour, regardless of their widely different optimal temperatures, suggests that thermostability does not directly relate to catalytic efficiency. The biophysical differences between monomeric DHFRs and TmDHFR are likely derived from the dimeric nature of the hyperthermophilic enzyme. An artificial dimeric variant of EcDHFR, Xet-3, was prepared by introducing residues at the dimer interface of TmDHFR. While thermostability of this variant is enhanced, it showed a great decrease in its steady-state and pre-steady-state rate constants. Given that the corresponding rate constants did not increase when the loops are released in the monomeric variant of TmDHFR, the lowered catalytic ability in Xet-3 is likely a consequence of geometric distortion of the active site and loss of loop flexibility that is catalytically important in EcDHFR. In contrast, the relatively poor activity of TmDHFR is not simply a consequence of reduced loop flexibility; the dimer interface of TmDHFR plays a rather complicated role in catalysis.
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Bevan, A. W. "Specificity of inhibitor binding to dihydrofolate reductase." Thesis, University College London (University of London), 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352532.
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Singh, Priyanka. "Enzyme catalysis and dynamics in dihydrofolate reductase." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5635.
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Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate bindings, chemical conversions, and products release. The relationship between protein motions and catalytic activity is of considerable contemporary interest in enzymology. To understand the factors influencing the rates of enzyme catalyzed reactions, the dynamics of the protein-solvent-substrate complex must be considered. The enzyme dihydrofolate reductase from Escherichia coli (EcDHFR) is often used as a model system in various biophysical studies, including those aimed at the examination of motions across the protein that may affect the catalyzed chemical transformation. Previously, molecular dynamics calculations, bioinformatics studies and intrinsic kinetic isotope effects (KIEs) of DHFR have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. This thesis describes studies that extend upon those initial results by studying the nature and extent of enzyme dynamics and motions in DHFR, both by using traditional experimental methods and by developing new biophysical probes of protein dynamics in this system. Kinetic techniques, site directed mutagenesis, methods involving isotopic labeling of substrates and proteins, immobilization techniques and molecular recognition force spectroscopy were combined to study EcDHFR. The major experimental methodology described in the subsequent chapters is the determination and analysis of intrinsic KIEs in a variety of EcDHFR mutants. The studies demonstrated that residues G121, M42 and F125, all of which are remote from the active site, FR participate in a network of coupled motions across the enzyme. Until recently, the missing link in our understanding of DHFR catalysis was the lack of a path by which such remote residues can affect the catalyzed chemistry at the active site. A later chapter describes studies that indicate synergism between a residue, G121, in that remote dynamic network and an active site residue, I14. In another related study all carbons and nitrogens, as well as non-exchangeable protons in EcDHFR were changed to their heavy isotopes (13C, 15N, 2H). This heavy enzyme has also been shown to be an efficient tool in addressing the heated debate regarding the role of protein dynamics in catalysis. Such enzyme generates a vibrationally perturbed "heavy ecDHFR", and the effect on the altered vibrations on catalysis was studied. Finally, the last two chapters describe techniques developed to immobilize DHFR on an AFM-mica chip via DNA linkers, concentration and activity assays of the immobilized enzyme, and single-molecule studies of the interaction between a tight inhibitor (methotrexate) and the enzyme. These studies reveal the distribution of states and interactions with ligands - a property not accessible for studies of a large ensemble of molecules. The high spatial and force resolution provided by AFM under physiological conditions have been utilized in this study to quantify the force-distance relationships of DHFR-methotrexate interactions. In the future, such an understanding of the interplay between enzyme function, structure and dynamics could eventually permit improved de novo construction of artificial biocatalysts, enable better inhibitor and drug design, and in general, further advance our ability to manipulate enzyme catalysis to our ultimate benefit.
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Al-Batayneh, Khalid Mubarak. "Mutations in Drosophila dihydrofolate reductase and methotrexate resistance." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63399.pdf.
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Khaw, Lake Ee. "Isotopic labelling of dihydrofolate reductase for NMR studies." Thesis, Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/25179.
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Tey, Lai-Hock. "Studies of site-selective glycosylation of dihydrofolate reductase." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55798/.
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Glycosylation is important for many molecular processes, although how glycosylation contributes to glycoprotein structure and function is not entirely clear. Other than that, the study of many of these events is complicated by the fact that natural glycoproteins normally occur as mixtures of glycoforms, therefore the isolation or synthesis of homogenous glycoproteins is an important task. Previous studies on deglycosylated proteins have shown that glycosylation reduces their catalytic activity and increase thermal stability. Therefore, we hypothesize that similar effects may be observed in the naturally unglycosylated dihydrofolate reductase from Escherichia coli (EcDHFR). Four different surface-exposed sites for the incorporation of a single cysteine residue were selected based on the protein crystal structure, which may or may not affect its dynamics. Homogeneous glycoproteins have been synthesized via chemoselective ligation of a glycosyl haloacetammide with the thiol of a cysteine, to produce site- selectively glycosylated forms of EcDHFR. Techniques such as mass spectrometry, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, ultraviolet (UV) visible spectroscopy and stopped-flow spectrophotometry have been used to identify and study the physical properties of different glycoforms of DHFR. Although there were some changes of the kinetic activity of the mutants of EcDHFR. the values were comparable to those of the wild-type protein. Interestingly, in four of the five cases studied. EcDHFRDM D87C, there was an increase in thermal stability upon site-selective glycosylation. The other mutants showed no effect. With the exception of the effect seen for the thermal stability of the D87C mutant, this is not in accordance with the original hypothesis. This suggest that the effect seen in the D87C mutant may be due to specific interactions of the carbohydrate moiety at certain points on the protein. An increase in resistance to thermal denaturation observed for proteins in sugar solutions may therefore also be due to binding of the sugars to specific sites on the protein. In conclusion, an effective method for the synthesis of homogeneous glycosylated and non-glycosylated proteins has been developed and applied to the site selective glycosylation of EcDHFR. The results also suggested that the kinetic properties of EcDHFR are not significantly affected by glycosylation. It may be the large effects in terms of protein stability which due to glycosylation only occur in naturally glycosylated proteins, and not in the naturally unglycosylated EcDHFR.
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Gibson, Marc William. "Characterisation of Trypanosoma brucei dihydrofolate reductase-thymidylate synthase." Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521705.
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Pang, Jiayun. "Computational studies of dihydrofolate reductase from Thermotoga maritima." Thesis, University of Birmingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433745.
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Sen, Arundhuti. "Explorations in enzymology: investigating dynamics in dihydrofolate reductase." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/2768.
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The relationship between enzyme dynamics and enzymatic catalysis has become a central topic in modern enzymology, and studies in this area promise to enrich our current understanding of catalysis in biological systems. Escherichia coli dihydrofolate reductase (EcDHFR) has been a frequent subject of study in the context of protein dynamics, due to its small size, biological ubiquity, and the fact that its structural, kinetic and mechanistic characteristics are well established. Intrinsic kinetic isotope effects (KIEs) have proven to be highly sensitive probes of the role of dynamics in EcDHFR catalyzed reaction, as they circumvent the kinetic complexity of the enzyme-catalyzed reactions, and extract information directly pertaining to the chemical step. Previously, studies of their temperature-dependence were used to probe the effect of mutations at residues distant from the active site upon the hydride-transfer reaction catalyzed by EcDHFR. The results of these experiments supported the presence of a network of residues that were dynamically linked to the hydride-transfer step, and were in excellent agreement with computational studies predicting the presence of such a network. This thesis aims to extend upon these results to study the nature and extent of the dynamic network in EcDHFR, both by using an established experimental methods and by developing new biophysical probes of protein dynamics in this system. The major experimental methodology utilized in the following chapters is the determination and analysis of KIEs in a variety of EcDHFR mutants. To facilitate these measurements, new synthetic routes to a range of isotopically labeled nicotinamide cofactors have been developed. Some of the labeled materials have been used to establish a sensitive, triple-isotope technique to competitively measure deuterium isotope effects in enzyme-catalyzed reactions in EcDHFR. Synthesized materials were usd to measure the temperature dependence of intrinsic KIEs in selected dynamically altered mutants of EcDHFR, viz. W133F and F125M DHFR. Crystal structures have been obtained for both these mutants as well as for the previously studied G121V isozyme, and the combination of kinetic and structural information discussed in the context of catalytically important dynamic fluctuations in EcDHFR. Pressure-dependence of deuterium KIEs is also developed as a tool to probe the role of dynamics and tunneling in the EcDHFR reaction, with the ultimate aim of establishing high-pressure KIE measurements as a complementary method to variable temperature measurements. Finally, molecular recognition force spectroscopy (MRFS) measurements of an EcDHFR self-assembled monolayer (SAM) on gold are described. The surprisingly active enzymatic SAM has been shown to be a promising platform for future MRFS experiments to measure the forces involved in EcDHFR dynamics. All together, these studies advanced our ability to study the role of enzyme dynamics and quantum tunneling in enhancing their chemistry.
15
RIMET, ODILE. "Etude de l'activite dihydrofolate reductase par spectrofluorimetrie : influence du cofacteur." Aix-Marseille 2, 1990. http://www.theses.fr/1990AIX22981.
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Behiry, Enas Mamdouh. "Dihydrofolate reductase and the physical basis of enzyme catalysis." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/56687/.
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Dihydrofolate reductase (DHFR) is the enzyme that catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH). The DHFR catalysed reaction has often been used to study enzymatic tunnelling and the contribution of protein dynamics to catalysis. To gain a better understanding of such phenomena and to investigate the key elements of structural adaptation in DHFR, in this thesis the hydride transfer reaction of DHFR from Moritella profunda (MpDHFR), a cold adapted enzyme, was studied and compared to the mesophilic and extensively studied enzyme from Escherichia coli (EcDHFR) and the thermophilic enzyme from Thermotoga maritima (TmDHFR). Chapter 1 gives a brief introduction to the thesis. Description of the materials and methods used in evaluating this work is reported in Chapter 2. In Chapter 3, the steady state and pre-steady state temperature dependences of the kinetic isotope effect (KIE) for the MpDHFR catalysed reaction was elevated, compared to data obtained for the mesophilic and the thermophilic DHFR homologues and the results interpreted according to the environmentally coupled tunnelling model. The work presented in Chapters 4 and 5 has investigated the role of dynamics during catalysis by DHFR using site directed mutagenesis. In Chapter 4, mutations were created in the GH loop for both EcDHFR and MpDHFR to elucidate the role of the occluded conformation during catalysis by DHFR. In Chapter 5, different MpDHFR and EcDHFR variants in the highly mobile M20 loop were generated and their temperature dependences of KIE were studied in addition to studying the two variants MpDHFR-G123V and MpDHFR-D124N in the catalytically important FG loop. The results obtained suggest that MpDHFR does not undergo the dynamical loop movements that have been recognized previously for EcDHFR in spite of following the same catalytic cycle. Further findings were found which contradict the current models that relate protein dynamics to catalysis efficiency, thus modifying these models has become essential. Chapter 6 has focused on studying the effect of different denaturants/salt concentrations on MpDHFR chemical step. Finally, a summary of the work presented in this thesis and future guidelines are provided in Chapter 7.
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Yap, Chee H. "Potential inhibitors of dihydrofolate reductase: synthesis and NMR spectroscopy." Thesis, Aston University, 1985. http://publications.aston.ac.uk/14508/.
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SICA, LUCAS. "Application de la microcalorimetrie aux mesures d'activte enzymatique : exemple de la dihydrofolate reductase." Aix-Marseille 2, 1988. http://www.theses.fr/1988AIX22953.
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Basran, Jaswir. "The role of conserved residues in Lactobacillus casei dihydrofolate reductase." Thesis, University of Leicester, 1994. http://hdl.handle.net/2381/35262.
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Mutants of three conserved residues in Dihydrofolate reductase (DHFR) from Lactobacillus casei have been studied to assess their roles in ligand binding and catalysis. Aspartate26 has been widely postulated to be the source of the proton in the reaction catalysed by DHFR; this was probed by the Aspartate26/Asparagine (D26N) mutation. The rate of hydride ion transfer, which governs kcat in the D26N mutant, is reduced by a factor of 10 (pH 7.5) when compared to the wild-type enzyme. The results argue against the role of Aspartate26 as the primary proton donor, but may be more consistent with a mechanism whereby it promotes enolisation of the substrate during the reaction. The Tryptophan21/Histidine (W21H) mutant binds the coenzyme NADPH over 1000-fold more weakly than wild-type DHFR. The magnitude of the negative cooperative effect between NADPH and FH4 (a crucial feature of the kinetic scheme of DHFR), has been greatly reduced in the W21H mutant, suggesting an important role for Tryptophan21 in the mechanism of negative cooperativity. The pH dependence of kcat for W21H (which is equivalent to the rate of hydride ion transfer) has a form that reflects the cooperative ionisation of two groups. Kinetic and NMR results suggest that the new Histidine21 is one of the groups responsible for the unusual ionisation curve. Substrate, inhibitor and coenzyme binding are unaffected by the Arginine57/Lysine (R57K) mutation. With the wild-type enzyme, loss of the ion-pair interaction between MTX and Arginine57 also leads to a loss of the interaction with Histidine28; this is not the case with the R57K mutant. The Arginine57/Lysine substitution has little effect on the rate of catalysis although the apparent pKa of kcat is reduced by 0.6 units, despite the site of catalysis being more than 15A away from the site of the mutation. The origin of this effect may be due to electrostatic or structural factors.
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Sasso-Bitran, Sophie. "Etude thermodynamique de la selectivite des inhibiteurs de la dihydrofolate reductase." Aix-Marseille 2, 1995. http://www.theses.fr/1995AIX22954.
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Thomas, Janette Ann. "Structure and activity studies on mutants of the enzyme dihydrofolate reductase." Thesis, University of Leicester, 1990. http://hdl.handle.net/2381/35171.
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During the course of this thesis four mutants of Lactobacillus casei dihydrofolate reductase have been studied to determine the role of the individual amino acids in the structure and function of the enzyme. The mutants tryptophan-21→ histidine and threonine-63 → alanine were successfully made by site specific mutagenesis. The mutant aspartate-26 →glutamine was successfully expressed and purified but did not bind as tightly to the methotrexate affinity column as the wild type. This mutant had only 1% of the activity of the wild type enzyme. The threonine-63 →glutamine mutant was successfully expressed and purified. The equilibrium binding constants of substrates and inhibitors binding to binary and ternary enzyme complexes were the same as wild type except the binding of the oxidised coenzyme to the mutant apoenzyme, which was reduced three fold. kcat, and Km for NADPH were unaffected by the mutation, however the apparent pKa of catalysis and the pKa of the isotope effect were both reduced by 0.5 pH units. Nmr analysis revealed no structural changes at residues near the mutation or to the bound coenzyme, however there were small structural affects seen at the substrate binding site, at least 15A away from the mutation. A movement along the backbone of helix-C, from glutamine-43 at the mutation site, to the substrate binding site is hypothesized to cause these affects. Expression of threonine-63 → alanine was only detected when the temperature was at 30°C rather than the usual 40°C. kcat and the binding of substrate and inhibitors were the same as wild type. The binding of coenzyme was reduced by six hundred fold, the rate of association reduced by fifty fold and the rate of dissociation increased by twelve fold. Nmr analysis revealed the adenine and the 2' phosphate moieties of NADPH are affected by the mutation, the adenine ring being further away from residues in the protein. These affects were not simply due to the loss of the hydrogen bond between threonine and the protein but must also be caused by some conformational change in the protein.
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Alfaraj, Rihaf. "Development of selective inhibitors of dihydrofolate reductase (DHFR) of Mycobacterium tuberculosis." Thesis, University of Bath, 2013. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669020.
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Mycobacterium tuberculosis is the causative organism for one of the pandemic diseases in the world, tuberculosis (TB). The length of treatment often results in multi-drug resistance (MDR) and patient non-compliance. One of the most important enzymes as a drug target for tuberculosis is dihydrofolate reductase (DHFR), which plays an important role in the folate cycle and inhibition of the enzyme stops cell growth. DHFR inhibitors are usually 2,4-diaminopyrimidines, which have high binding affinity to the enzyme but have the potential to inhibit the human enzyme. This project focuses on the development of new inhibitors with improved potency and selectivity for the M. tuberculosis enzyme. Inhibitors containing a 5-phenyl group were targeted in order to increase lipophilicity and binding to the enzyme, whilst reducing binding to the human enzyme. Condensation of diethyl phenylmalonate with guanidine followed by chlorination and amination of the carbonyl group gave 2-amino-6-chloro-4-pmethoxybenzylamino- 5-phenylpyrimidine, which was deprotected to give 6-chloro-2,4- diamino-5-phenylpyrimidine. Reaction of 2-amino-6-chloro-4-p-methoxybenzylamino- 5-phenyl-pyrimdine with different aromatic and aliphatic amines was also investigated. Amination with an amino alcohol in presence of potassium carbonate in the absence of solvents was used to synthesise a number of analogues. Deprotection of the pmethoxybenzylamine was achieved by DDQ oxidation to give the desired 2,4-diamino- 5-phenyl-6-aminoalcohol-pyrimidine products. The synthesis of the triol motif began with protection of ribonolactone and reduction of the carbonyl group to give the diol. Derivatisation of the product with various protecting goups was investigated.
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Li, Jiayue. "The preservation of protein dynamics from bacteria to human dihydrofolate reductase." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6984.
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Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR.
Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites.
As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.
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Kolmodin, Karin. "Computer Simulation of Protein Tyrosine Phosphatase Reaction Mechanisms and Dihydrofolate Reductase Inhibition." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : [Univ.-bibl. [distributör]], 2001. http://publications.uu.se/theses/91-554-5148-9/.
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Graffner, Nordberg Malin. "Approaches to soft drug analogues of dihydrofolate reductase inhibitors : Design and synthesis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5017-2/.
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Wooden, Jason. "Analysis of resistance to inhibitors of Plasmodium falciparum dihydrofolate reductase in yeast /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/10261.
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Povinelli, Christine Marie. "Genetic analysis of the dihydrofolate reductase and thymidylate synthase genes of bacteriophage T4." Diss., Georgia Institute of Technology, 1987. http://hdl.handle.net/1853/25347.
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Rooke, Stuart. "Some reactions of vinyl sulfimides and synthesis of novel inhibitors of dihydrofolate reductase." Thesis, University of Warwick, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263814.
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Tang, Lei. "The determination and structural analysis of site-directed dihydrofolate reductase and insulin mutants." Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338337.
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Maurer, Barry James. "Dihydrofolate reductase gene amplification in human cell lines VA2-B and hela BU25." Diss., Pasadena, Calif. : California Institute of Technology, 1995. http://resolver.caltech.edu/CaltechETD:etd-10232007-082738.
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Hastings, Michele Dawn. "Analysis of dihydrofolate reductase variations in relation to antifolate resistance in Plasmodium vivax /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/10269.
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Yang, Qing X. "Nuclear magnetic resonance studies on the structure and dynamics of dihydrofolate reductase and its ligands." Diss., Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/30856.
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DAM, JULIE. "Etudes experimentales et theoriques des interactions proteine-proteine au sein de la dihydrofolate reductase r67." Paris 7, 2000. http://www.theses.fr/2000PA077055.
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Dans le but d'affiner notre comprehension de la physique a la base des interactions proteiques, l'assemblage en tetramere de la dhfr r67 a ete etudiee par une approche multidisciplinaire. L'interface d'association a ete modifiee par mutagenese dirigee, les effets des mutations sur la constante d'equilibre de dissociation (k d) mesures experimentalement et calcules par modelisation moleculaire, et enfin, l'effet des mutations sur la structure a ete controle par cristallographie aux rayons x. L'association dimere-dimere est dependante du ph, du fait d'une liaison hydrogene entre la ser 59 d'un dimere et la leu 62 d'un autre dimere. Des mutations simples introduites sur ces positions ont conduit a des dimeres inactifs incapables de s'associer en tetramere. Nous avons exploite cette incapacite d'auto-association pour identifier des paires de dimeres modifies, capables de complementer. Une analyse combinatoire a permis d'identifier une dizaine d'heterotetrameres. Une nouvelle methode, appelee titration en iso-fluorescence, a ete concue pour mesurer des k d avec precision. Il a ete montre qu'un echange de monomeres se produisait au sein des heterotetrameres, mais que pour certaines paires, ce phenomene etait trop lent pour etre significatif. Deux modeles d'association avec ou sans echange ont ete etablis pour l'analyse des donnees. Cette methode, minimisant les erreurs experimentales, permet de determiner des k d avec des incertitudes de 3 a 30% selon les conditions. La confrontation de ces donnees experimentales avec les resultats de calcul theoriques montre un bon accord. Les calculs ont suggere une explication pour le moteur de l'association de certains mutants. Ils ont souligne l'importance energetique de la relaxation structurale due aux mutations. Cette relaxation a ete controlee par cristallographie. Ces premiers calculs suggerent que les effets de mutation sur l'association peuvent etre predits, et qu'a ce jour la physique des proteines semble bien comprise.
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Adrian, Peter V. "Trimethoprim-resistant dihydrofolate reductase genes in South African isolates of aerobic Gram-negative commensal faecal flora." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/26335.
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A survey was conducted in South Africa to determine the incidence of resistance in aerobic Gram-negative commensal faecal flora. Faecal specimens from 272 out of 362 (75%) healthy volunteers carried trimethoprim-resistant strains, from which 357 trimethoprim-resistant organisms were isolated. Trimethoprim resistance was transferable by conjugation in 55% of the isolates. The majority of the isolates were resistant to other antimicrobial agents including ampicillin 71.4% and tetracycline 88%. Most of these resistance phenotypes co-transferred with trimethoprim resistance. Analysis of 148 plamids revealed 79 different restriction profiles which indicated that there is a large gene pool of trimethoprim-resistant organisms in the faecal flora. High-level resistance to trimethoprim (MIC ≥ 1024mg/l) occurred in 98.6% of the isolates suggesting that resistance in these isolates was mediated by the production of additional trimethoprim resistant dihydrofolate reductase (DHFR) genes. To determine the epidemiology of these genes, oligonucleotide probes were designed from the nucleotide sequence of a heterogeneous region which occurs within all trimethoprim resistant DHFR genes. Hybridisation experiments revealed that contrary to all previous data, the most prevalent DHFR of the transferable genes which hybridised was the type Ib (30%), followed by the type VIII (23%), V (13%), Ia (6%), VII (3%) and XII (0.5%). On the other hand the type VII, (38%) was the most prevalent dihydrofolate reductase gene in the 161 (45%) isolates which did not transfer their resistance factors, followed by type Ia (25%), type Ib (12%), type V (2%) and type VIII (0.5%).
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Mariam, Nakintu. "Detection of mutations in dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) in Plasmodium falciparum in eastern Sudan." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-155295.
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Stojkovic, Vanja. "Contribution of active site dynamics to enzyme catalysis: study on a series of mutants of dihydrofolate reductase." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/5062.
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This thesis describes an effort to expand current knowledge of catalysis in biological systems. The focus is on understanding how enzymes activate covalent bonds and specifically to study C-H bond activation via enzymes. The work presented here examined the role of protein dynamics and hydrogen tunneling in enzyme catalysis. Dihydrofolate reductase from Escherichia coli (ecDHFR), which catalyzes a single hydride transfer reaction, was selected as the model system for these studies. Intrinsic kinetic isotope effects (KIEs) have been shown to be highly sensitive probes in examining the chemical steps of enzymatic reactions, especially since they can indirectly infer the role of certain dynamic fluctuations in the ecDHFR-catalyzed reaction. This study provides evidence in support of the phenomenological Marcus-like model presently well accepted amongst both experimental enzymologists and some members of the computational community — this model suggests that certain molecular fluctuations prevail during the enzyme-catalyzed hydrogen transfer reaction and assist the chemical step. Previous studies of the temperature-dependence of KIEs focused on examining the network of residues that are dynamically linked to the hydride-transfer step and are localized far from the active site; a network initially proposed by computational studies. This thesis, on the other hand, focuses on the effect of the active site environment on the C-H→C transfer. While no spectroscopy experiments were performed to measure the dynamics in the active site, sensitive kinetic experiments were used to examine the physical features of the C-H→C transfer via rigorous perturbation of the donor-acceptor-distance (DAD). KIE measurements on a series of carefully designed active site mutants have been interpreted using a Marcus-like model and complemented by results obtained via molecular dynamic simulations and X-ray crystallography. Active site mutants were designed to alter the DAD and its dynamics in a controlled manner with a minimal effect on the active-site electrostatics. The results suggest that the mutations have affected the reorganization energy necessary for the system to reach the transition state and have modulated the average DAD as well as its distribution at the transition state. The study on the active-site mutants was extended on N23PP—a dynamically altered mutant that was the source of an extensive debate in the field due to opposing views regarding the altered dynamics and its role in assisting the hydride transfer step. Findings presented in this thesis indicate that temperature dependent kinetic complexity masked the intrinsic KIEs in the earlier studies, and that our methodology revealed the significant differences between the natures of the hydride transfer catalyzed by the WT and by the dynamically impaired mutant. Collectively these results further our understanding of the role of enzyme dynamics and quantum tunneling in enhancing enzymatic reactions. In the future these results will be correlated to findings from vibrational spectroscopy, high-level calculations and NMR studies (as they become available) in order to establish the structure-dynamic-function-relationship both in ecDHFR and in enzymes in general.
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Duboué-Dijon, Elise. "Interactions entre une biomolécule et son environnement : de la dynamique d'hydratation à la catalyse enzymatique." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066314/document.
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Les biomolécules sont naturellement immergées dans l’eau, qui joue un rôle clé dans de nombreux processus biologiques. Réciproquement, les propriétés de l’eau sont affectées par la présence de la biomolécule. Dans cette thèse, nous combinons modèles théoriques et simulations numériques pour obtenir une description à l’échelle moléculaire des interactions entre une biomolécule et son environnement. Le manuscrit est structuré en deux parties, abordant deux aspects complémentaires de cette interaction complexe. La première partie est consacrée à la perturbation induite par une biomolécule sur l’eau. Nous déterminons en quoi la couche d’hydratation diffère de l’eau bulk et identifions les facteurs moléculaires en jeu. Nous comparons ensuite les couches d’hydratation d’une protéine antigel et d’une protéine modèle afin de déterminer si les propriétés d’hydratation peuvent expliquer l’activité antigel. Nous étudions enfin la dynamique d’hydratation de l’ADN. Nous obtenons une image résolue spatialement des propriétés de sa couche d’hydratation et y caractérisons les différentes sources d’hétérogénéité. La deuxième partie s’intéresse au rôle de l’environnement sur la catalyse enzymatique. Nous étudions deux systèmes distincts, avec des questions différentes mais une même méthodologie. Nous examinons d’abord le rôle de résidus dans le site actif de la dihydrofolate réductase et obtenons une interprétation moléculaire de résultats expérimentaux récents. Enfin, nous nous intéressons à la catalyse enzymatique en solvant organique, où l’addition de petites quantités d’eau permet d’accélérer la réaction. Nous recherchons une description à l’échelle moléculaire de cet effetBiomolecules are immersed in an aqueous solvent, which plays a key role in a wide range of biochemical processes. In addition, the properties of water molecules in the hydration shell are perturbed by the presence of the biomolecule. In this thesis, we combine theoretical models and numerical simulations to provide a molecular description of the interplay between a biomolecule and its environment. The manuscript is structured in two parts, addressing two complementary aspects of this complex interaction. In the first part we focus on the perturbation induced by a biomolecule on water molecules. We determine how much the hydration shell differs from bulk water and we identify the molecular factors at play. We then compare the hydration shells of an antifreeze protein and of a typical protein and investigate whether the shell structure and dynamics can explain the antifreeze properties. We finally study the hydration dynamics of a DNA dodecamer where slow water dynamics was suggested. We obtain a spatially resolved picture of DNA hydration and investigate the sources of heterogeneity. In the second part we examine the role of the environment in the chemical step of enzyme catalysis. We focus on two distinct systems with different questions, but relying on a common simulation methodology. We first examine the role of specific active site residues in catalysis by dihydrofolate reductase and we provide a molecular interpretation of recent experimental results. We finally study the role of water in enzyme catalysis in organic solvents, where addition of small amounts of water was shown to accelerate the chemical step. We seek a molecular scale description of this effect
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Solé, Ferré Anna. "Correction of point mutations at the endogenous locus of the mammalian dihydrofolate reductase gene using polypurine reverse hoogsteen hairpins." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/379822.
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This work is focused on the study of Polypurine Reverse Hoogsteen (PPRH) hairpins and their ability as gene correction tools.
The repair of a point mutation at its endogenous gene locus has been the focus of many researchers during the past decades by using various approaches such as gene replacement, gene augmentation therapy (GAT) and different repair oligonucleotides. Cystic fibrosis, sickle-cell anemia and Tay-Sachs disease are some examples of the high number of disorders caused by a single-point mutation. In this work we present an alternative repair methodology using PPRH molecules, that although first developed in our laboratory as a gene-silencing tool, we hypothesized that they could also be applied as a gene correction tool.
PPRHs are double-stranded DNA molecules formed by two antiparallel homopurine domains linked by a 5-thymidine loop, which form intramolecular reverse Hoogsteen bonds. PPRHs have been described to effectively bind to the dsDNA forming a triplex structure (Coma et al., 2005), and have been designed against either the template or the coding strand of the dsDNA (de Almagro et al., 2009, de Almagro et al., 2011a).
Taking advantage of the ability of PPRHs to form a triplex structure with the pyrimidine strand of the dsDNA, in the present work we wanted to explore the ability of PPRHs to correct point mutations. First of all, we studied the in vitro conditions for PPRHs to bind to dsDNA and to maintain it in an open conformation by binding assays. Then, we designed different repair-PPRHs by adding to the PPRH core a repair tail complementary to the mutated region of the DNA except for the nucleotide to be corrected. These repair-PPRHs were used in mammalian cells to repair a point mutation in a dihydrofolate reductase (dhfr) plasmid.
Finally, those results led us to further demonstrate the correction capability of repair-PPRHs in different mutant cell lines containing single point mutations in the endogenous dhfr gene. In addition, we developed improved Long-distance-repair¬PPRHs (LDR-PPRHs) to correct mutations that are very distant with respect to the DNA target where the PPRH core binds.
Considering the possible high proportion of guanines in a PPRH sequence, an in vitro characterization of these molecules was carried out to study the effect of these guanines in the formation of secondary structures, such as G-quadruplex. The triplex conformation formed by repair-PPRHs with their pyrimidine target sequences was also studied even when repair-PPRHs folded in a G-quadruplex structure instead of a hairpin.
As a second part of this thesis, we developed a new application of the electrophoretic mobility shift assay (EMSA) technique, to demonstrate the binding between miRNAs and their target sequences. For over two decades, research in our laboratory has been focused on the pharmacogenomic study of methotrexate (MTX) resistance in cancer chemotherapy upon inhibition of DHFR. Beside gene amplification, our research group has shown that many genes are involved in this mechanism. In this direction, cellular genes and micro-RNAs such as miR-224 and its target genes SLC4A4, CDS2 and HSPC159 were identified to play an important role in the resistance to MTX in colon cancer cells (Mencia et al., 2011). For this reason, miR-224 was chosen to show in a direct and specific manner, its ability to bind to the SLC4A4 target gene, thus setting up the EMSA as a simple and useful method to validate the interaction between a miRNA and a specific mRNA target.Aquest treball es centra en l'estudi de pinces de polipurines "polypurine reverse Hoogsteen (PPRH)", i la seva capacitat com eina en la correcció de gens. La reparació d'una mutació puntual en el seu locus endogen ha sigut el principal tema de molts investigadors en les últimes dècades, utilitzant diverses estratègies com per exemple la teràpia de substitució de gens (GAT) i diferents oligonucleòtids de reparació. La fibrosi quística, anèmia de cèl.lules falciformes i la malaltia de Tay-Sachs son alguns exemples de la gran quantitat de trastorns causats per una mutació puntual. En aquest treball, presentem una metodologia de reparació alternativa que utilitza molècules PPRH, desenvolupades en el nostre laboratori inicialment com a eina de silenciament gènic, amb la hipòtesi que també es podrien aplicar com a eina de correcció de gens. Els PPRH son unes molècules d'ADN de doble cadena, formades per dos dominis de homopurines antiparal•eles, connectades per un bucle de 5 timidines, que formen enllaços de Hoogsteen reversos i intramoleculars. Els PPRHs s'han descrit per unir-se eficaçment a la doble cadena d'ADN formant una estructura de tríplex, i han sigut dissenyats tant contra la cadena motlle com contra la cadena codificant de l'ADN. Tenint en compte la capacitat dels PPRHs per formar una estructura de tríplex amb la cadena de pirimidines de l'ADN, en l'estudi presentat aquí volíem explorar la capacitat dels PPRHs per corregir mutacions puntuals. En primer lloc, es van estudiar les condicions in vitro en les que els PPRHs s'unien a la doble cadena de l'ADN i la mantenien oberta, mitjançant assajos d'unió. Tot seguit, hem dissenyat diferents PPRHs reparadors, tot afegint, al nucli del PPRH, una cua reparadora, complementaria a la regió mutada de l'ADN excepte pel nucleòtid que ha de ser corregit. Aquests PPRHs reparadors s'han utilitzat en cèl.lules de mamífer per reparar una mutació puntual en un plàsmid de la dihydrofolate reductasa (dhfr). Per últim, els resultats positius ens van portar a estudiar la capacitat dels PPRHs reparadors per reparar diferents línies cel.lulars que contenien diferents tipus de mutacions puntuals en el gen endogen de la dhfr. A més, també hem desenvolupat un PPRH millorat, anomenat LDR-PPRH, per "Long-Distance-Repair-PPRH", per reparar mutacions puntuals molt distants respecte la seqüència diana del nucli del PPRH. Tenint en compte la possible alta proporció de guanines en una seqüència de PPRH, es va dur a terme una caracterització in vitro d'aquestes molècules per estudiar l'efecte de les guanines en la formació d'estructures secundàries, com per exemple els G-quàdruplex. També es va estudiar la conformació de triple hèlix formada pels PPRH reparadors amb les seves seqüències diana de pirimidines, fins i tot quan el PPRH reparador es plega en una estructura de G-quàdruplex enlloc de en forma de pinça. Com a segona part de la tesi, hem desenvolupat una nova aplicació de la tècnica d'assaig de canvi de mobilitat electroforètica (EMSA), per demostrar la unció entre els miRNAs i les seves seqüències diana. Durant més de dues dècades, la investigació en el nostre laboratori s'ha centrat en l'estudi farmacogenòmic de la resistència al metotrexat (MTX) en la quimioteràpia contra el càncer, per la inhibició del gen de la dhfr. A més de l'amplificació gènica, el nostre grup ha demostrat que molts gens estan involucrats en el mecanisme, així com també hi estan involucrats els micro-RNAs. El miR-224 i els seus gens diana, SLC4A4, CDS2 i HSPC159 van ser identificats per jugar un paper important en la resistència al MTX en cèl.lules de càncer de colon. Per aquesta raó, el miR-224 va ser l'escollit per mostrar d'una manera directa i específica, la seva habilitat per unir-se al gen diana SLC4A4, establint així la tècnica d'EMSA com un mètode senzill i útil per validar la interacció entre un miRNA i la seva diana específica de mRNA.
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Groom, Colin Roger. "Crystallographic studies on the binding of inhibitors to wild-type and site-directed mutants of a mouse dihydrofolate reductase." Thesis, University of Leeds, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397792.
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Tahar, Rachida. "Etude de la dihydrofolate reductase de plasmodium falciparum et plasmodium vivax, bases moleculaires et biochimiques de la resistance aux antifoliniques." Paris 12, 1999. http://www.theses.fr/1999PA120054.
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L'emploi massif des antifoliniques pour le traitement du paludisme dans le passe a permis l'emergence de souches de p. Falciparum et de p. Vivax resistantes a ces medicaments. Les antifoliniques sont des analogues du substrat de la dihydrofolate reductase-thymidylate synthase (dhfr-ts), enzyme bifonctionnelle impliquee dans la biosynthese des folates. Des etudes anterieures ont mis en evidence la correlation entre des mutations ponctuelles dans le domaine de la dhfr et la resistance in vivo et in vitro a la pyrimethamine et au cycloguanil chez p. Falciparum. Chez p. Vivax, la resistance aux antifoliniques n'avait pas ete etudiee in vitro. Nous avons isole le gene dhfr-ts de p. Vivax par pcr a partir de l'adn genomique utilisant des oligonucleotides degeneres. L'analyse de la sequence montre une structure en trois regions (dhfr, pont, ts) comparable a celle des autres plasmodiums. Le sequencage du gene de 30 isolats de zones geographiques diverses montre que le domaine ts est hautement conserve, cependant que le domaine dhfr, plus polymorphe, possede des mutations ponctuelles et un tandem repetitif (ggdn) de longueur variable. Trois mutations ponctuelles ser59arg, ser117asn et ile173leu dans la dhfr de p. Vivax sont homologues des mutations cys58arg, ser108asn et ileu164le de la dhfr de p. Falciparum impliquees dans la resistance a la pyrimethamine et au cycloguanil. Nous avons fait exprimer les enzymes recombinantes sauvage dhfr s e r 5 9 s e r 1 1 7 et mutante dhfr a r g 5 9 a s n 1 1 7 dans e. Coli. Apres isolement et renaturation de ces enzymes, l'etude enzymatique in vitro a montre que les mutations ponctuelles ser58arg et ser117asn dans le domaine de la dhfr augmentent nettement les constantes d'inhibition (ki) et la ci 5 0 vis-a-vis des antifoliniques (pyrimethamine, cycloguanil, trimethoprime et methotrexate). Etant donne le role cle chez p. Vivax du codon 117 dans la resistance aux antifoliniques, nous avons mis au point un test moleculaire rapide permettant a la fois de diagnostiquer l'espece plasmodiale et de determiner la nature du codon 117. Nous avons aussi essaye de definir les bases moleculaires de la resistance in vivo de p. Falciparum a l'association des antifoliniques et des antifoliniques qui agissent en synergie sur la meme voie enzymatique. La resistance a la pyrimethamine chez les isolats portant des mutations au niveau de la dhfr peut etre compensee partiellement par l'effet synergique de l'interaction pyrimethamine-sulfadoxine. Des echecs therapeutiques ont ete cependant constates chez des patients infectes par des souches de p. Falciparum ayant un genotype triple mutant ser108asn, asn51ile et cys59arg dans le domaine dhfr et un genotype sauvage ou portant la mutation ala437gly dans la dhps.
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Hankins, Eleanor Gray. "Drug resistance in Plasmodium falciparum : the role of point mutations in dihydropteroate synthase and dihydrofolate reductase analyzed in a yeast model /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/10290.
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Eldin, de Pecoulas Philippe. ""Plasmodium falciparum" et "Plasmodium vivax" : Etude comparée de la dihydrofolate réductase-thymidylate synthétase, polymorphisme moléculaire et relations avec la chimiosensibilité aux antifoliniques." Paris 5, 1996. http://www.theses.fr/1996PA05P621.
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Corrêa, Denis da Silva. "Docking de compostos da família das ariloxazinas em enzimas relacionadas com a malária." Universidade Federal de São Carlos, 2010. https://repositorio.ufscar.br/handle/ufscar/6964.
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Malaria disease, caused mainly by Plasmodium falciparum parasite, afflicts about 500 million people and causes nearly one million deaths every year. For the development of new drugs against this disease, one possible approach is to identify an enzyme that plays a key role in P. falciparum development and presents significantly different properties from the corresponding human one. These differences can be exploited in the design of specific inhibitors of the parasite s protein, thus, three different enzymes were selected as possible targets. As there are evidences suggesting that increasing oxidative stress can effectively inhibit the growth of the malarial parasite the enzyme Glutathione Reductase of P. falciparum (PfGR), responsible for the parasite s antioxidant defense, has become a potential target for the design and development of inhibitors. The second target was the P. falciparum Dihydrofolate Reductase-Thymidylate Synthase (PfDHFR-TS), and in this case blocking its action stops the dTMP production and DNA synthesis in the parasite. The third chosen target was the P. falciparum Lactate Dehydrogenase (PfLDH), whose inhibition interrupts the ATP formation and thus causing the death of the parasite. So that a family of arilloxazines compounds, together with chloroquine and methylene blue, were studied by means of docking simulations in the binding sites of these enzymes and also in the corresponding human enzymes for comparison. The three-dimensional structures of the enzymes and of chloroquine and methylene blue were obtained from the Protein Data Bank (PDB). The structures of the arilloxazines compounds, in turn, were obtained by molecular modeling with HyperChem 6.01 and MOPAC2009 programs, using as starting models similar crystallographic structures deposited in the Cambridge Structural Database. Docking simulations were performed using GOLD 4.0.1. The docking results showed that the enzymes PfGR and PfDHFR-TS are not the preferential targets of chloroquine. For the methylene blue it was possible to elucidate its binding mode in hGR and PfGR. Regarding the arilloxazines it was possible to show that they present their higher affinity for hGR, followed by PfGR, hDHFR, PfDHFR-TS, PfLDH and hLDH. In the case of GRs, the interface site was the preferred binding site. The results suggest that if arilloxazines compounds with higher affinity for PfGR are desirable then a pentafluorophenyl should be attached at the N10 position, as in the 2e compound. When searching for arilloxazines with higher affinity for PfLDH, it seems to be desirable a carboxymethyl group at the N3 position (as in 5b) and a pentafluorophenyl group at N10 (as in 2e). Finally, the results suggest that in general the studied arilloxazines probably will present a higher affinity for hDHFR than PfDHFR-TS. All these results are an important starting point for the design of new arilloxazines ligands so that they can be used as lead compounds in the search for new drugs against malaria.
A malária, causada principalmente pelo Plasmodium falciparum, atinge cerca de 500 milhões de pessoas e causa aproximadamente um milhão de mortes todos os anos. Para o desenvolvimento de novos fármacos contra esta doença, uma das abordagens possível é identificar uma enzima que desempenhe papel vital no desenvolvimento do P. falciparum e apresente propriedades significantemente diferentes das enzimas humanas correspondentes, de modo que tais diferenças possam ser exploradas no design de inibidores específicos à proteína do parasita. Existem evidências sugerindo que aumentar o estresse oxidativo pode inibir eficientemente o crescimento do parasita causador da malária e, portanto, a enzima Glutationa Redutase do P. falciparum (GRPf), responsável por sua defesa antioxidante, tornou-se um alvo em potencial para o desenvolvimento de inibidores. Também, o bloqueio da ação da Diidrofolato Redutase-Timidilato Sintase do P. falciparum (DHFR-TSPf) interrompe a produção de dTMP e a síntese de DNA no parasita. Ainda, espera-se que a inibição da Lactato Desidrogenase do P. falciparum (LDHPf) interrompa a produção de ATP no parasita e, consequentemente, cause sua morte. Portanto, estudou-se o comportamento de compostos da família das ariloxazinas, da cloroquina e do azul de metileno nos sítios de ligação destas enzimas, além das enzimas humanas correspondentes para fins de comparação, por meio de cálculos de docking. As estruturas tridimensionais das enzimas foram obtidas no Protein Data Bank (PDB). As estruturas dos inibidores da família das ariloxazinas, por sua vez, foram obtidas por meio de modelagem molecular, utilizando os programas HyperChem 6.01 e MOPAC2009, a partir de estruturas cristalográficas semelhantes obtidas no Cambridge Structural Database; já as estruturas da cloroquina e do azul de metileno foram obtidas também no PDB. Os cálculos de docking destes compostos nos sítios de ligação das enzimas estudadas foram realizados utilizando o programa GOLD 4.0.1. Com base nos resultados de docking, sugere-se que as enzimas GRPf e DHFR-TSPf não são alvos preferenciais da cloroquina. Também, pôde-se elucidar o possível modo de ligação do azul de metileno nas enzimas GRh e GRPf. No geral, foi possível sugerir ainda que as ariloxazinas devam apresentar maior afinidade pela GRh, seguida por GRPf, DHFRh, DHFR-TSPf, LDHPf e LDHh, nesta ordem. Nas GRs, o sítio da interface foi o sítio preferencial de ligação. Para se buscar inibidores da família das ariloxazinas com maior afinidade pela GRPf, sugere-se considerar um pentafluorfenil como substituinte na posição N10, como no composto 2e. Ainda, na busca por ariloxazinas com maior afinidade pela LDHPf, sugere-se considerar um carboximetil na posição N3 (como o de 5b) e um pentafluorfenil na posição N10 (como em 2e). Por fim, foi obtido que as ariloxazinas estudadas possivelmente apresentarão, em geral, uma maior afinidade pela DHFRh do que pela DHFR-TSPf. Estes dados podem ser tomados como ponto de partida para o design de novos compostos da família das ariloxazinas, a fim de que possam atuar como compostos líderes na busca por novos fármacos contra a malária.
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Karatzas, Antonios. "Evaluation of chemoprotection conferred against alkylating drugs by a bicistronic retroviral vector expressing the A3 isoform of glutathione S-transferase and a variant of human dihydrofolate reductase." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80298.
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The principal dose-limiting toxicity of most chemotherapeutic agents is myelosuppression. In this study we evaluated the chemoprotection conferred against alkylators by a bicistronic retroviral vector expressing the chemoresistance genes GSTA3 and DHFRL22Y. As reported herein, GID-transduced murine hematopoietic cells exhibited greater survival compared to control cells in presence of chlorambucil concentrations ranging from 5 x 10-6 M to 1 x 10-4M. After increasing the fraction of GID-expressing cells via chemoselection with TMTX and NBMPR, the level of resistance was increased by up to 2.9-fold at 5 x 10-5 M chlorambucil. GID-transduced hematopoietic cell enrichment by ex vivo chemoselection was limited, under our experimental conditions, by a marked loss of hematopoietic reconstitution ability, while in vivo chemoselection was shown to hold greater potential to selectively enrich GID-expressing blood cells. The presence of replication competent virus in the experiments makes it difficult to reach to conclusive interpretations of results. Notwithstanding the unfortunate outcome of murine leukemia that did not allow for the evaluation of GID-mediated hematopoietic chemoprotection in vivo, the promising in vitro experimental results reported here warrant further investigation of this approach.
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Alonso, Hernan, and hernan alonso@anu edu au. "Computer Modelling and Simulations of Enzymes and their Mechanisms." The Australian National University. The John Curtin School of Medical Research, 2006. http://thesis.anu.edu.au./public/adt-ANU20061212.161155.
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Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr).
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The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrBDHFNADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB.
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Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a minimalistic enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues.
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MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism.
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Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.
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Legrand, Baptiste. "Analyse d'interactions moléculaires par RMN : Étude de la DHFR en présence d'osmolytes et structures de pseudopeptides antimicrobiens en environnement." Phd thesis, Rennes 1, 2009. https://tel.archives-ouvertes.fr/tel-00453405.
Full textAbstract:
Les osmolytes sont des solutés accumulés dans les cellules de nombreux organismes lors d'une contrainte hyperosmotique pour maintenir le volume cellulaire. Ils peuvent néanmoins induire une inhibition des enzymes dont les bases moléculaires sont au centre de nombreuses études. Nous avons étudié la DHFR, en présence de divers solutés. Les osmolytes ne modifient pas la structure globale de la DHFR et inhibent son activité tout en stabilisant sa structure. Cette inhibition est liée à la diminution du k[indice :]off du produit en présence d'osmolytes. L'étude de la dynamique interne de la DHFR apporte des réponses sur l'origine de l'inhibition de la DHFR en présence d'osmolytes. Une seconde partie présente nos travaux sur la relation structure-activité de peptides antimicrobiens. Ce projet s'inscrit dans la course au développement de nouvelles molécules actives pour substituer les antibiotiques conventionnels. Nous avons déterminé les structures de peptides en environnement membranaire modèleThe osmolytes are small molecules accumulated by cells of a wide variety of organisms in response to hyperosmotic stress to maintain the cellular volume. Nervetheless, enzyme activity is inhibited by these cosolutes and the molecular basis of their effects on the proteins properties is of great interest. We studied the DHFR in presence of the osmolytes. We demonstrate that its overall structure is maintained. While the osmolytes stabilize the DHFR structure, they inhibit its activity at the same time. The k[index :]off, of substrate analogues decreases with increasing the osmolyte concentrations and reflects the variation of DHFR catalytic rate. The study of the DHFR dynamic on several timescales gives answer of the origins of the DHFR inhibition in presence of osmolytes. The second chapter concerns the study of the structure-activity relationship of antimicrobial peptides. The main objective of this project is to develop new drugs. We solved NMR solution structure of in various model membranes
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Yahashiri, Atsushi. "Comparative investigations of H-transfer in dihydrofolate reductases from different families." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/763.
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This thesis presents an effort to understand the C-H-C transfer in enzymatic reactions from the comparison of different variants of enzymes that have unrelated protein sequences and structures, but catalyze the same chemical transformation. I evaluated the kinetic isotope effects (KIEs) and their temperature dependences and interpreted the findings in accordance with Marcus-like models. The enzyme system studied is dihydrofolate reductase (DHFR), which catalyzes the reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) using reduced β-nicotinamide adenine dinucleotide 2' phosphate (NADPH) as a reducing agent. H-transfer reactions in typical enzymes from three genetically unrelated families, E. coli chromosomal DHFR (cDHFR, FolA), plasmid coded R67 DHFR (FolB), and pteridine reductase 1 (PTR1, FolM) were comparatively investigated. Chapter I provides a brief introduction to the thesis. Chapter II presents optimized procedures for a one-pot, enzymatic microscale synthesis of several NADPH isotopologues used in KIE experiments. Chapter III focuses on the application of novel competitive primary H/D KIE determinations. Chapter IV compares the H-transfer reactions between primitive R67 DHFR and the chromosomal DHFR, and Chapter V describes the investigation of H-transfer reactions at high and low ionic strengths with theoretical and experimental approaches in order to understand the unusual enhancement in H-transfer rate of R67 DHFR with increasing ionic strength. Chapter VI discusses an improved PTR1 purification procedure and comparisons of steady state kinetic parameters using PTR1 and cDHFR with H2F and dihydrobiopterin (H2B) substrates. Thus, the investigation of the H-transfer reaction catalyzed by cDHFR with an unnatural substrate, H2B is described. Finally, a summary is provided and future directions are discussed in Chapter VII.
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Martins, João Paulo Machado. "Triagem virtual de inibidores da enzima di-hidrofolato redutase de Schistosoma mansoni (SmDHFR)." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/9/9138/tde-18102017-152832/.
Full textAbstract:
A esquistossomose é uma das principais causas de morbidade em países Tropicais e Subtropicais, gerando graves consequências socioeconômicas. Atualmente, os fármacos disponíveis para o tratamento da desta doença são praziquantel e oxamniquina, porém relatos de baixa susceptibilidade do parasita a esses medicamentos sugerem a necessidade de novas estratégias terapêuticas para o tratamento da doença. Todavia, existe pouco interesse da indústria farmacêutica no desenvolvimento de fármacos contra doenças tropicais e negligenciadas, entre as quais se encontra a esquistossomose. Devido a estes fatores, o presente trabalho teve por objetivo geral utilizar ferramentas computacionais para identificar inibidores da SmDHFR candidatos a novos fármacos. Avaliou-se as características exclusivas para a proteína de S. mansoni por meio de uma análise das sequências FASTA em comparação com a DHFR de outros organismos. A fim de garantir a ação seletiva dessas moléculas frente a enzima do parasita, os campos moleculares de interação seletivos para SmDHFR foram calculados e empregados na construção do modelo farmacofórico, o qual foi utilizado na triagem virtual de inibidores de SmDHFR. Os estudos computacionais realizados nos permitiram a seleção de 20 moléculas com uma boa complementariedade com o modelo farmacofórico gerado e com potencial para serem inibidores de SmDHFR.Schistosomiasis is one of morbidity\'s main causes in tropical and subtropical countries, which leads to serious socioeconomic consequences. Praziquantel and oxamniquina are the drugs currently available for treating this disease, but reports points that the parasite has been resistant to both drugs, which suggests the need for new therapeutic strategies for the treatment of this disease. However, there is little interest in the pharmaceutical industry in developing drugs against neglected tropical diseases, including schistosomiasis. Due to these factors, the present work has the general objective to use computational tools to identify SmDHFR inhibitors which could be good candidates for developing new drugs. Evaluation of the exclusive characteristics of the S. mansoni protein were performed by FASTA sequence analyses in comparison to DHFR from other organisms. In order to guarantee the selective action of these molecules against the parasite enzyme, the molecular interaction fields selective for SmDHFR were calculated and used in the construction of the pharmacophoric model, which was further used in the virtual screening of SmDHFR inhibitors. Computational studies were performed and those led us to 20 molecules with a good complementarity with the pharmacophoric model that was previously generated and with potential to be SmDHFR inhibitors.
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Tu, Yongxue. "Etude physicochimique et biochimique des dihydrofolates reductases et de l'effet antifolate des thiosemicarbazones." Paris 7, 1988. http://www.theses.fr/1988PA077164.
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Thomson, Christopher J. "Biochemical analysis of the recent plasmid-encoded trimethoprim-resistant dihydrofolate reductases in gram-negative bacteria." Thesis, University of Edinburgh, 1990. http://hdl.handle.net/1842/19351.
Full textAbstract:
The most important mechanism of trimethoprim (Tp) resistance is the plasmid-encoded production of an additional Tp resistant dihydrofolate reductase (DHFR). The epidemiology of plasmid-mediated resistance to Tp has been studied by biochemical typing of the enzymes responsible. In recent years several new Tp resistant DHFRs have been identified. The type III enzyme, considered the rarest of the plasmid-mediated DHRFs was isolated once in New Zealand in 1979 and never subsequently detected. However the biochemical properties of a Tp resistant DHFR isolated in Nottingham were examined as DNA gene probing had suggested that the enzyme was a type III and the biochemical properties confirmed this. The enzymes responsible for Tp resistance in two outbreaks of Shigella in the United States were examined. Detailed biochemical analysis suggested that the two enzymes were different from each other but similar to the type III. Therefore the three enzymes were subsequently renamed types IIIa, IIIb and IIIc. The properties of the type IIIb enzyme were very similar to the original type IIIa; however, sequence analysis of the N-terminal of this protein showed that it was quite distinct from the type IIIa. The type IV DHFR was isolated in South India in 1984 and is the only inducible plasmid-mediated DHFR. Examination of the induction process suggested that the resistance mechanism of the enzyme was similar to chromosomal hyperproduction where resistance is achieved not because of the insensitivity of the DHFR, but because it is produced in such an amount that it 'swamps' the inhibiting Tp. Purification and sequence analysis of the type IV DHFR revealed that the enzyme was similar to the chromosomal DHFR and that it was complexed with NS1 an E.coli DNA binding protein. The biochemical properties of the type V DHFR, which was isolated in Sri Lanka in 1985, were similar to those of the type 1 enzyme, with the exception that the type V has an unusually low molecular mass when measured in Sephadex. On native polyacrylamide gel electrophoresis however the two enzymes co-migrate, this biochemical similarity suggests the two enzymes are closely related. The efficiency of plasmid-mediated resistance to Tp has compromised the use of this drug in many parts of the world, from bichemical studies it is clear that plasmid-mediated enzymes continue to evolve.