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Friemann, Rosmarie. "Structure-function studies of iron-sulfur enzyme systems /." Uppsala : Dept. of Molecular Biology, Swedish Univ. of Agricultural Sciences, 2005. http://epsilon.slu.se/a504.pdf.
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O'Neil, Crystal L. "Enzyme Exploitation: Manipulating Enzyme Function for Therapy, Synthesis and Natural Product Modification." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1293722936.
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Gwozd, Chantelle Sabrina. "The structural basis of ubiquitin conjugating enzyme function." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq22989.pdf.
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Friemann, Rosmarie. "Structure-function studies of iron-sulfur enzyme systems /." Uppsala : Dept. of Molecular Biology, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/a504-ab.html.
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Roden, D. L. "High specificity automatic function assignment for enzyme sequences." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1321566/.
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The number of protein sequences being deposited in databases is currently growing rapidly as a result of large-scale high throughput genome sequencing efforts. A large proportion of these sequences have no experimentally determined structure. Also, relatively few have high quality, specific, experimentally determined functions. Due to the time, cost and technical complexity of experimental procedures for the determination of protein function this situation is unlikely to change in the near future. Therefore, one of the major challenges for bioinformatics is the ability to automatically assign highly accurate, high-specificity functional information to these unknown protein sequences. As yet this problem has not been successfully solved to a level both acceptable in terms of detailed accuracy and reliability for use as a basis for detailed biological analysis on a genome wide, automated, high-throughput scale. This research thesis aims to address this shortfall through the provision and benchmarking of methods that can be used towards improving the accuracy of high-specificity protein function prediction from enzyme sequences. The datasets used in these studies are multiple alignments of evolutionarily related protein sequences, identified through the use of BLAST sequence database searches. Firstly, a number of non-standard amino acid substitution matrices were used to re-score the benchmark multiple sequence alignments. A subset of these matrices were shown to improve the accuracy of specific function annotation, when compared to both the original BLAST sequence similarity ordering and a random sequence selection model. Following this, two established methods for the identification of functional specificity determining amino acid residues (fSDRs) were used to identify regions within the aligned sequences that are functionally and phylogenetically informative. These localised sequence regions were then used to re-score the aligned sequences and provide an assessment of their ability to improve the specific functional annotation of the benchmark sequence sets. Finally, a machine learning approach (support vector machines) was followed to evaluate the possibility of identifying fSDRs, which improve the annotation accuracy, directly from alignments of closely related protein sequences without prior knowledge of their specific functional sub-types. The performance of this SVM based method was then assessed by applying it to the automatic functional assignment of a number of well studied classes of enzymes.
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Szeto, Michelle Wing Yan. "QM/MM studies of enzyme structure and function." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445894.
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Liou, Geoffrey. "Enzyme structure, function, and evolution in flavonoid biosynthesis." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122067.
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This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019<br>Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references.<br>Plant specialized metabolism is a key evolutionary adaptation that has enabled plants to migrate from water onto land and subsequently spread throughout terrestrial environments. Flavonoids are one particularly important class of plant specialized metabolites, playing a wide variety of roles in plant physiology including UV protection, pigmentation, and defense against herbivores and pathogens. Flavonoid diversity has increased in conjunction with land plant evolution over the past 470 million years. This dissertation examines the structure, function, and evolution of enzymes in the flavonoid biosynthetic pathway. First, we structurally and biochemically characterized orthologs of chalcone synthase (CHS), the enzyme that catalyzes the first step of flavonoid biosynthesis, from diverse plant lineages. By doing so, we gained insight into the sequence changes that gave rise to increased reactivity of the catalytic cysteine residue in CHS orthologs in euphyllophytes compared to basal land plants. We then developed methods and transgenic plant lines to study the in vivo function of these CHS orthologs, as well as whether their functional differences play a role in redox-based regulation of flavonoid biosynthesis. Finally, we examined enzymes involved in the biosynthesis of galloylated catechins, a highly enriched class of flavonoids in tea that are thought to have health benefits in humans. These findings contribute to an understanding of the evolution of enzyme structure and function in flavonoid biosynthesis, and how it has facilitated the adaptation of plants to a wide variety of terrestrial habitats.<br>by Geoffrey Liou.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology
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Ljungberg, Liza. "Angiotensin-converting enzyme in cardiovascular function and dysfunction." Doctoral thesis, Linköpings universitet, Fysiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-67215.
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Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system, converting angiotensin I to the vasoactive peptide angiotensin II, and degrading bradykinin. Angiotensin II is a multifunctional peptide, acting on a number of different tissues. A common genetic variation in the gene encoding ACE; ACE I/D polymorphism influences the level of ACE in the circulation, and has been linked to increased risk for cardiovascular disease. This thesis aimed to explore the connection between ACE and cardiovascular function and dysfunction. The impact of nicotine and nicotine metabolites on ACE in cultured human endothelial cells was studied. Nicotine as well as nicotine metabolites induced increased ACE activity in cultured human endothelial cells. In elderly men a higher ACE level was seen in smokers compared to non-smokers. Furthermore, diabetes was associated with higher circulating ACE. Increased ACE level may represent a cellular mechanism which contributes to vascular damage. Elderly men carrying the ACE D allele had higher abdominal aortic stiffness compared to men carrying the I/I genotype. Our data suggest that the mechanism by which the ACE D allele modulates aortic wall mechanics is independent of circulating ACE levels. Previous studies have indicated a link between the D allele and abdominal aortic aneurysm. Increased aortic stiffness suggests impaired vessel wall integrity, which combined with local hemodynamic and/or inflammatory factors may have a role in aneurysm formation. Subjects with left ventricular dysfunction had higher levels of circulating ACE compared to those with normal left ventricular function, while there was no association between ACE and central hemodynamics. ACE might play a role in the pathogenesis of left ventricular dysfunction and our findings suggest a direct effect on the heart rather than affecting central blood pressure.
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Khan, Amjad. "NMR spectroscopy studies of enzyme function and inhibition." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:698d69c7-d4f1-4bc7-bf0b-3b9e7fb3a4fe.
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The work described in this Thesis focuses on the application of NMR spectroscopy methods in understanding the function and inhibition of two protein systems; these are ?-butyrobetaine hydroxylase (BBOX) and the bacterial potassium ion efflux (Kef) system. BBOX belongs to the super family of enzymes called the 2- oxoglutarate (2OG) and FeII dependent oxygenase and is involved in the biosynthesis of L-carnitine in humans and other prokaryotes. BBOX is a current drug target for the treatment of myocardial infarction. Kef is a ligandgated system that protects bacteria from toxic electrophilic species. Kef is inhibited by the binding of cytoplasmic glutathione (GSH) to KTN (K+ transport and nucleotide) binding domains and activated by glutathione-S-conjugates (GS-X). Since bacterial Kef activation during electrophilic exposure is a critical determinant of their survival, perturbation of Kef activity is potentially a novel target for the development of antibiotic drugs. 1H NMR direct ligand-observation was employed to study the binding interaction of the natural substrate ?- butyrobetaine (GBB) and co-substrate 2OG with BBOX. A 1H NMR-based dual-reporter ligand displacement method was developed to assess the nature of inhibitor binding to BBOX i.e to determine whether an inhibitor competes with GBB or 2OG or both. The method was exemplified with a set of isoquinoline-based inhibitors; the results reveal 'cystallographically unexpected' structure-activity relationship with some inhibitors competing 2OG only and some competing both 2OG and GBB. Using 1H NMR spectroscopy, a simple and efficient BBOX inhibition assay was developed for inhibitor IC50 measurement. Similarly, 1H NMR-based assays were applied to demonstrate that the cation-p interaction between the substrates and aromatic cage residues of BBOX play a critical role in BBOX substrate recognition. 1H NMR spectroscopy was applied to show that in the absence of a 2OG oxygenase, ascorbate in the assay mixture is slowly degraded by the dissolved oxygen to yield H2O2 which simultaneously leads to 2OG breakdown into succinate. It is proposed that in the assays of 2OG oxygenases, the apparent increase in the level of "uncoupled" 2OG turnover with ascorbate over time could possibly be due to the artifacts of the ascorbate induced-2OG breakdown instead of being due to enzyme catalysis. Other reducing agents were also found to oxidise identically by the dissolved oxygen as ascorbate in the mixture and result in 2OG breakdown. In the Kef system, 1H NMR direct ligand-observation was applied to investigate the influence of each functional group of the Kef activating ligand glutathione-S-N-tertiary butylsuccinimide on its binding interaction (KD 0.4 μM) with Kef-QCTD (Q-linker carboxy terminal domain; a KTN domain) from Shewanella denitrificans (sd) with the aim of developing novel non-peptidic ligands (antibacterial agents) of Kef. In addition, 19F NMR was employed to develop an efficient ligand-observed binding assay for Kef that was used for ligand screening as well as measuring their binding dissociation constant value from a single NMR spectrum. Finally, a 1H NMR technique was applied to confirm that the electron density found in the nucleotide binding pocket in the crystal structure of apo-sdKef-QCTD is unambiguously an AMP molecule that is naturally bound to the protein and has a role in stabilising the dimeric form of KTN domains (Kef proteins).
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Hamilton, Russell S. "DAROGAN : enzyme function prediction from multiple sequence alignments." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/14972.
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The function of an enzyme is often dependent on a few key functional residues and the principal objective of this project was to develop a novel function prediction system which takes advantage of this by comparing the conserved amino acids in known enzyme families to those in a putative enzyme. Multiple sequence alignments of well characterised enzyme families (with an E.C. number assigned) are used to create unordered sets of conserved functional residues, termed <i>Treads</i>. Comparison of a query proteins <i>Tread </i> to the reference <i>Treads</i> is undertaken by projecting them in multidimensional space and measuring distance between them. A major advantage of this prediction strategy implemented in DAROGAN is that it should be able to recognise similarities in the functions of enzymes that are not similar in structure or sequence. The method has been tested with regard to its ability to predict cofactor-dependencies toward pyridoxal-5’-phosphate, thiamine, glutathione and folic acid utilising enzymes. An area of application for DAROGAN is the prediction of previously described enzyme functions in organisms with completed genomes to which no gene and protein sequence could be assigned though the standard annotation processes. Investigations were made into the potential of utilising the DAROGAN method to propose candidates for the missing pyridoxal-5’-phosphate utilising enzymes in the <i>E. coli</i> genome according to EcoCyc. Candidates are proposed by assessing the 511 sequences from the GeneQuiz project, to which there are homologues in other species, but with uncertain functions. The assessment takes the form of using the DAROGAN method to determine the similarities of each of the sequences to the reference <i>Treads.</i>
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Harris, Katharine Morse. "Studies of structure, function and mechanism in pyrimidine nucleotide biosynthesis." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2594.
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Thesis advisor: Evan R. Kantrowitz<br>Thesis advisor: Mary F. Roberts<br>Living organisms depend on enzymes for the synthesis using small molecule precursors of cellular building blocks. For example, the amino acid aspartate is synthesized in one step by the amination of oxaloacetate, an intermediate compound produced in the citric acid cycle, exclusively by means of an aminotransferase enzyme. Therefore, function of this aminotransferase is critical to produce the amino acid. In the Kantrowitz Lab, we seek to understand the molecular rational for the function of enzymes that control rates for the biosynthesis of cellular building blocks. If one imagines the above aspartate-synthesis example as a single running conveyer belt, any oxaloacetate that finds its way onto that belt will be chemically transformed to give aspartate. We can extend this notion of a conveyer belt to any enzyme. Therefore, the rate at which the belt moves dictates the rate of synthesis. Now imagine many, many conveyer belts lined in a row to give analogy to a biosynthesis pathway requiring more than one enzyme for complete chemical synthesis. This is such the case for the biosynthesis of nucleotides and glucose. Nature has developed clever tricks to exquisitely control the rate of product output but means of altering the rate of one or some of the belts in the line of many, without affecting the rate of others. This type of biosynthetic rate regulation is termed allostery. Studies described in this dissertation will address questions of allosteric processes and the chemistry performed by two entirely different enzymes and biosynthetic pathways. The first enzyme of interest is fructose-1,6-bisphosphatase (FBPase) and its role in the biosynthesis of glucose. Following FBPase introduction in Chapter One, Chapter Two describes the minimal atomic scaffold necessary in a new class of allosteric type 2 diabetes drug molecules to effect catalytic inhibition of Homo sapiens FBPase. Following, is the second enzyme of interest, aspartate transcarbamoylase (ATCase) and its role in the biosynthesis of pyrimidine nucleotides. Succeeding ATCase introduction in Chapter Three, Chapter Four describes a body of work exclusively about the catalysis by ATCase. This work was inspired by the human form of the enzyme following the human genome project completion providing data that show likely Homo sapiens ATCase is not allosterically regulated. Chapter Five describes work on a allosterically-regulated, mutant ATCase and provides a biochemical model for the molecular rational for the catalytic inhibition upon cytidine triphosphate (CTP) binding to the allosteric site. The experimental techniques used for answering research questions were enzyme X-ray crystallography, in silico docking, kinetic assay experiments, genetic sub-cloning and genetic mutation<br>Thesis (PhD) — Boston College, 2012<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Loftus, Katherine Marie. "Studies of the Structure and Function of E.coli Aspartate Transcarbamoylase." Thesis, Boston College, 2006. http://hdl.handle.net/2345/580.
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Thesis advisor: Evan R. Kantrowitz<br>E.coli Aspartate transcarbamoylase (ATCase) is the allosteric enzyme that catalyzes the committed step of the de novo pyrimidine biosynthesis pathway. ATCase facilitates the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The holoenzyme is a dodecamer, consisting of two trimers of catalytic chains, and three dimers of regulatory chains. ATCase is regulated homotropically by its substrates, and heterotropically by the nucleotides ATP, CTP, and UTP. These nucleotides bind to the regulatory chains, and alter the activity of the enzyme at the catalytic site. ATP activates the rate of ATCase's reaction, while CTP inhibits it. Additionally, UTP and CTP act together to inhibit the enzyme synergistically, each nucleotide enhancing the inhibitory effects of the other. Two classes of CTP binding sites have been observed, one class with a high affinity for CTP, and one with a low affinity. It has been theorized that the asymmetry of the binding sites is intrinsic to each of the three regulatory dimers. It has been hypothesized that the second observed class of CTP binding sites, are actually sites intended for UTP. To test this hypothesis, and to gain more information about heterotropic regulation of ATCase and signal transmission in allosteric enzymes, the construction of a hybrid regulatory dimer was proposed. In the successfully constructed hybrid, each of the three regulatory dimers in ATCase would contain one regulatory chain with compromised nucleotide binding. This project reports several attempts at constructing the proposed hybrid, but ultimately the hybrid enzyme was not attained. This project also reports preliminary work on the characterization of the catalytic chain mutant D141A. This residue is conserved in ATCase over a wide array of species, and thus was mutated in order to ascertain its significance<br>Thesis (BS) — Boston College, 2006<br>Submitted to: Boston College. College of Arts and Sciences<br>Discipline: Chemistry<br>Discipline: College Honors Program
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Brokx, Stephen John. "Structure and function of enzyme I of the PTS." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0029/NQ63848.pdf.
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van, der Merwe Mariè. "Enzyme architecture and flexibility affect DNA topoisomerase I function." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/ABSTRACTS/2007-026-van_der_Merwe-Index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.<br>Title from title page screen (viewed on July 29, 2008). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xiii, 175 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 161-175).
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Chiang, Ranyee Agnes. "Ligand-based perspectives on the evolution of enzyme function." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324594.
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Alderson, Rosanna Grace. "Tracking the evolution of function in diverse enzyme superfamilies." Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/10496.
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Tracking the evolution of function in enzyme superfamilies is key in understanding how important biological functions and mechanisms have evolved. New genes are being sequenced at a rate that far surpasses the ability of characterization by wet-lab techniques. Moreover, bioinformatics allows for the use of methods not amenable to wet lab experimentation. We now face a situation in which we are aware of the existence of many gene families but are ignorant of what they do and how they function. Even for families with many structurally and functionally characterized members, the prediction of function of ancestral sequences can be used to elucidate past patterns of evolution and highlight likely future trajectories. In this thesis, we apply in silico structure and function methods to predict the functions of protein sequences from two diverse superfamily case studies. In the first, the metallo-β-lactamase superfamily, many members have been structurally and functionally characterised. In this work, we asked how many times the same function has independently evolved in the same superfamily using ancestral sequence reconstruction, homology modelling and alignment to catalytic templates. We found that in only 5% of evolutionary scenarios assessed, was there evidence of a lactam hydrolysing ancestor. This could be taken as strong evidence that metallo-β-lactamase function has evolved independently on multiple occasions. This finding has important implications for predicting the evolution of antibiotic resistance in this protein fold. However, as discussed, the interpretation of this statistic is not clear-cut. In the second case study, we analysed protein sequences of the DUF-62 superfamily. In contrast to the metallo-β-lactmase superfamily, very few members of this superfamily have been structurally and functionally characterised. We used the analysis of alignment, gene context, species tree reconciliation and comparison of the rates of evolution to ask if other functions or cellular roles might exist in this family other than the ones already established. We find that multiple lines of evidence present a compelling case for the evolution of different functions within the Archaea, and propose possible cellular interactions and roles for members of this enzyme family.
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Rees, D. "Studies on energy metabolism by phosphorous nuclear magnetic resonance." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370295.
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Charnock, Simon James. "Structure/function analysis of a family 10 glycosol hydrolase." Thesis, University of Newcastle upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262920.
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Carrasco, Rodríguez Patricia. "Study of the physiological function of carnitine palmitoyltransferase 1C enzyme." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/79172.
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Carnitine palmitoyl transferase 1 (CPT1) enzymes catalyze the conversion of long-chain acyl-CoA to acyl-carnitines, thus facilitating the entry of long-chain fatty acids to the mitochondria, where they undergo β-oxidation. There are three isoforms: the liver isoform CPT1A (Esser, V. 1993), the muscle isoform CPT1B (Yamazaki, N. 1995) and the brain-specific isoform CPT1C (Price, N. 2002). CPT1A and CPT1B are localized in the outer mitochondrial membrane and are rate-limiting enzymes in fatty-acid β-oxidation.
The CPT1C isoform, was first described in 2002, is expressed exclusively in the central nervous system, with a homogeneous distribution in all areas such as hippocampus, cortex, hypothalamus, cerebellum and others. CPT1C enzyme highly differs from the two other isozymes. Its C-terminal region is longer than that of the other CPTs (Price, N. 2002). It is located in the endoplasmic reticulum (ER) of cells, rather than in mitochondria, and so it does not facilitate fatty acid oxidation (Sierra, A.Y. 2008). Analysis of amino sequence of CPT1C reveals that all important residues for CPT1 activity are conserved in CPT1C enzyme, despite this, no catalytic activity was found (Price, N. 2002; Wolfgang, M.J. 2006), but it binds the CPT1 physiological inhibitor malonyl-CoA with the same affinity as CPT1A (Wolfgang, M.J. 2006). Finally, CPT1C is only present in mammals and appears to stem from a relatively recent CPT1A gene duplication (Price, N. 2002). The other isozymes are expressed in such organisms as fish, reptiles, amphibians or insects. This suggests a specific role for CPT1C in more evolved brains.
At the physiological level, CPT1C contributes to the control of food intake and energy homeostasis (Wolfgang, M.J. 2006; Gao, X.F. 2009). Two independent groups developed a CPT1C-KO mouse, and both lines showed decreased food intake respect to wild-type animals (WT). However, when fed a high-fat diet, they were more susceptible to obesity and diabetes, presenting lower rates of peripheral fatty acid oxidation. All these effects were attributed to the hypothalamic function of CPT1C, since ectopic over-expression of CPT1C in hypothalamus protected mice from adverse weight gain caused by high-fat diet (Dai, Y. 2007). Moreover, the involvement of CPT1C in energy homeostasis has also been confirmed in transgenic animals over-expressing CPT1C specifically in brain (Reamy, A.A. 2011). At the molecular level, in collaboration with the group of Dr. Gary Lopaschuk, we showed that CPT1C is involved in the anorectic action of leptin, by modulating ceramide synthesis in the arcuate nucleus (ARC) of the hypothalamus (Gao, S. 2011).
Interestingly, recent findings in tumor cells showed a new, unexpected role of CPT1C in the metabolic transformations reported in tumor cell growth (Zaugg, K. 2011). The authors demonstrated that CPT1C is frequently expressed in human lung tumors and protects cancerous cells from death induced by glucose deprivation or hypoxia. The results suggest that CPT1C might provide unidentified fatty-acid derived products that would be beneficial for cell survival under metabolic stress.
However, despite these recent findings about CPT1C, little is known about its catalytic activity or its physiological function in other brain areas.
We demonstrate that CPT1C has low CPT1 activity although it has similar affinity for its substrates: carnitine and palmitoyl-CoA than CPT1A isoform. The present study also shows that CPT1-KO mice have reduced long-chain acyl-carnitine levels in the hippocampus, hypothalamus or cerebellum.
We examined whether CPT1C is expressed in the peripheral nervous system: in the ventral horn of the spinal cord (motor neurons) and in the sensitive ganglions, in addition to the brain. We found that CPT1C is expressed in both regions, albeit at lower levels than in the brain. We also examined CPT1C expression along mouse development, and we found that CPT1C protein expression is present in early stage of embryos at day 15, is increased postnatally and reaches its expression peak in adulthood.
Moreover, CPT1C is expressed in pyramidal neurons of hippocampus and is located in ER throughout the neuron, even inside dendritic spines. We used molecular, cellular and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C over-expression in primary hippocampal cultured neurons increased ceramide levels, an effect that was blocked by treatment with myriocin, an inhibitor of the de novo synthesis of ceramide. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in hippocampus, cerebellum, striatum and motor cortex, mainly during fasting. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic over-expression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition and motor function, we performed the hippocampus-dependent Morris Water Maze (MWM) test and some motor tests on mice. Results show that CPT1C-KO mice are hypoactive and exhibit clear deficits in motor function, especially in coordination skills and strength. Moreover, CPT1C deficiency strongly impairs spatial learning without affecting memory or cognitive flexibility. So, all these results demonstrate that CPT1C regulates the de novo synthesis of ceramide in ER of hippocampal neurons and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.<br>ESTUDIO DE LA FUNCIÓN FISIOLÓGICA DE LA ENZIMA CPT1C
La isoforma carnitina palmitoil transferasa 1C (CPT1C) se expresa únicamente en cerebro y ha sido implicada en la regulación hipotalámica de la ingesta de alimentos y la homeostasis energética. No obstante, su función molecular y su papel en otras áreas del cerebro son desconocidas. Hemos demostrado que CPT1C se expresa en las neuronas piramidales del hipocampo y se localiza en el retículo endoplásmico a lo largo de la neurona, incluso dentro de las espinas dendríticas. Hemos utilizado métodos moleculares, celulares y conductuales para determinar la función de CPT1C. En primer lugar, analizamos la implicación de CPT1C en el metabolismo de la ceramida. La sobre-expresión de CPT1C en neuronas de hipocampo aumentó los niveles de ceramidas, un efecto que fue bloqueado por el tratamiento con miriocina, un inhibidor de la síntesis de novo de la ceramida. En consecuencia, los ratones CPT1C knock-out (CPT1C-KO) demostraron una reducción de los niveles de ceramidas en el hipocampo, cerebelo, estriado y corteza motora principalmente durante el ayuno. A nivel celular, la deficiencia en CPT1C afecta a la morfología de las espinas dendríticas mediante el aumento de filopodios inmaduros y reduciendo el número de espinas maduras. La densidad de protrusiones totales o el área de la cabeza de la espinas dendrítica no se vieron afectadas. El tratamiento de las neuronas en cultivo con ceramida exógena, como la sobre-expresión ectópica de CPT1C, revirtieron el fenotipo de las espinas CPT1C-KO, lo que indica que CPT1C regula la maduración de las espinas dendríticas a través de las ceramidas. Para estudiar las repercusiones del fenotipo CPT1C-KO en la cognición y en la habilidad motora se realizaron diferentes test conductuales. Los resultados del test cognitivo demostraron que la deficiencia de CPT1C perjudica al aprendizaje espacial. Por otra parte, la realización de test motores demostraron que los ratones CPT1C son hipoactivos y tienen disminuida tanto la coordinación motora como la fuerza muscular. Todos estos resultados demuestran que CPT1C regula la síntesis de novo de ceramidas en el retículo endoplásmico de las neuronas y éste es un mecanismo necesario para la correcta maduración de las espinas dendríticas y para el adecuado procedimiento del aprendizaje espacial y la función motora.
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Luu, Luong. "Structure/function studies of hP450RAI, a retinoic acid metabolizing enzyme." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ36051.pdf.
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De, Ferrari Luna Luciana. "On combining collaborative and automated curation for enzyme function prediction." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/7538.
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Data generation has vastly exceeded manual annotation in several areas of astronomy, biology, economy, geology, medicine and physics. At the same time, a public community of experts and hobbyists has developed around some of these disciplines thanks to open, editable web resources such as wikis and public annotation challenges. In this thesis I investigate under which conditions a combination of collaborative and automated curation could complete annotation tasks unattainable by human curators alone. My exemplar curation process is taken from the molecular biology domain: the association all existing enzymes (proteins catalysing a chemical reaction) with their function. Assigning enzymatic function to the proteins in a genome is the first essential problem of metabolic reconstruction, important for biology, medicine, industrial production and environmental studies. In the protein database UniProt, only 3% of the records are currently manually curated and only 60% of the 17 million recorded proteins have some functional annotation, including enzymatic annotation. The proteins in UniProt represent only about 380,000 animal species (2,000 of which have completely sequenced genomes) out of the estimated millions of species existing on earth. The enzyme annotation task already applies to millions of entries and this number is bound to increase rapidly as sequencing efforts intensify. To guide my analysis I first develop a basic model of collaborative curation and evaluate it against molecular biology knowledge bases. The analysis highlights a surprising similarity between open and closed annotation environments on metrics usually connected with “democracy” of content. I then develop and evaluate a method to enhance enzyme function annotation using machine learning which demonstrates very high accuracy, recall and precision and the capacity to scale to millions of enzyme instances. This method needs only a protein sequence as input and is thus widely applicable to genomic and metagenomic analysis. The last phase of the work uses active and guided learning to bring together collaborative and automatic curation. In active learning a machine learning algorithm suggests to the human curators which entry should be annotated next. This strategy has the potential to coordinate and reduce the amount of manual curation while improving classification performance and reducing the number of training instances needed. This work demonstrates the benefits of combining classic machine learning and guided learning to improve the quantity and quality of enzymatic knowledge and to bring us closer to the goal of annotating all existing enzymes.
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Chang, Cheng-Fu. "Structure-function and regulation studies of angiotensin-converting enzyme 2." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3122.
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Sharma, Narayan Prasad. "STRUCTURE/FUNCTION STUDIES ON METALLO-B- LACTAMASE ImiS FROM Aeromonas bv. sobria." Oxford, Ohio : Miami University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1181583976.
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Stewart, Lorna. "The active transport systems of proline and potassium in Escherichia coli." Thesis, University of Aberdeen, 1987. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU006337.
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The transport systems for proline and potassium represent two of the active transport systems in Escherichia coli. They have further similarities that their transport may be utilized as a response to osmotic perturbations in the environment. The exact mechanism of transport had not been totally elucidated. The transport of proline had been assumed to operate as a proton symport and as such had been used as a model system when other transport systems were being investigated. This study has demonstrated that the major route of proline uptake through the proline permease 1 (PP1), operates as a Na+ - proline cotransport which may accept Li+ in the place of Na+. Unusually, Na+ stimulates the Vmax of transport with little or no effect on the Km. In addition to this transport system, there are two other proline uptake systems which function primarily for the transport of betaine. The transport of K + is also facilitated by more than one system. The Kdp system is a K+ transporting ATPase; the TrkF system is a low rate transport system which may represent leak through another pathway. The TrkA transport system is the major system but the mechanism is not known. Transport through the system is energised by ATP and a pmf, while exchange through the system requires only ATP. The role of ATP was investigated in this study by the use of metabolic inhibitors and vesicles. It was determined that the availability of ATP affected the steady state level of potassium in the cells rather than the rate of potassium upake. It was speculated that ATP would act as a regulator of the system which would be driven by the pmf. ATP may regulate TrkA through phosphorylation or by allosteric modification of the carrier.
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Tuuttila, Ari. "Structure and function of MMP-2 and its inhibitor TIMP-2 /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4468-7/.
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Wen, Bo. "Analysis of human CYP3A4 structure-function relationships using photoaffinity labels /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8154.
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Lombardi, Olivia. "Investigating the role of mRNA capping enzyme in C-MYC function." Thesis, University of Dundee, 2017. https://discovery.dundee.ac.uk/en/studentTheses/4816aeec-c481-4494-9a07-56e74a83c08e.
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C-MYC is a transcription factor and a potent driver of many human cancers. In addition to regulating transcription, C-MYC promotes formation of the mRNA cap which is important for transcript maturation and translation. However, the mechanistic details of C-MYC-dependent mRNA capping are not fully understood. Since anti-cancer strategies to directly target the C-MYC protein have had limited success, enzymatic co-factors or effectors of C-MYC present attractive alternatives for therapeutic intervention of C-MYC-driven cancers. mRNA capping enzyme (CE) initiates mRNA cap formation by catalysing the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide. The involvement of CE in C-MYC-dependent mRNA capping and C-MYC function has not yet been explored. Therefore, I sought to determine whether C-MYC regulates CE, and whether CE is required for C-MYC function. I found that C-MYC promotes CE recruitment to RNA polymerase II (RNA pol II) transcription complexes and to regions proximal to transcription start sites on chromatin. Consistently, C-MYC increases RNA pol II-associated CE activity. Interestingly, cells driven by C-MYC are highly dependent on CE for C-MYC-induced target gene expression and cell transformation, but only when C-MYC is overexpressed; C-MYC-independent cells or cells retaining normal control of C-MYC expression are insensitive to CE inhibition. C-MYC expression is also dependent on CE. Taken together, I present a bidirectional regulatory relationship between C-MYC and CE which is potentially therapeutically relevant. Studies here strongly suggest that inhibiting CE is an attractive strategy to selectively target cancer cells which have acquired deregulated C-MYC.
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Webb, Catherine Marie. "Polychlorinated biphenyl effects on avian hepatic enzyme induction and thyroid function." Thesis, Virginia Tech, 2006. http://hdl.handle.net/10919/33915.
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Polychlorinated biphenyls (PCBs) decrease thyroid function in rats and mice by inducing activity of a liver enzyme, uridine diphosphate-glucuronosyltransferase (UDP-GT), thereby increasing thyroxine (T4) clearance. This loss of T4 can lead to hypothyroidism. In this study, an assay was validated for measuring UDP-GT activity toward T4 in Japanese quail (Coturnix japonica). Then UDP-GT induction by Aroclor 1254 was evaluated in quail, and quail and mice were compared in their responses to Aroclor 1254. In Experiment 1, Japanese quail and Balb/c mice were dosed orally with vehicle or Aroclor 1254 (250 or 500 mg/kg) and sacrificed five days later. In Experiment 2, Japanese quail were dosed orally with vehicle or Aroclor 1254 (500 mg/kg) and sacrificed either five or 21 days later. Total liver UDP-GT capacity increased with Aroclor 1254 exposure in all treatment groups of both species. Enzyme induction led to a trend to decreased plasma T4 concentrations at both doses and exposure times in quail and significantly decreased plasma T4 concentrations at both doses in mice. PCBs altered thyroid function in quail, but they did not become hypothyroid. This was in contrast to mice, which did become hypothyroid. It is unclear how PCBs affect the hypothalamic-pituitary-thyroid (HPT) axis in quail, and activation of the HPT axis appears to be inhibited in mice. Overall, quail showed a lesser response than mice to equivalent doses of Aroclor 1254, so it appears that birds may be less vulnerable to PCBs than mammals.<br>Master of Science
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Redelinghuys, Pierre. "Structure-function relationship of angiotensin-converting enzyme : glycosylation and domain-selectivity." Doctoral thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/3147.
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Raines, C. A. "Comparison of the effects of trypsin and chymotrypsin on thylakoid membrane function." Thesis, University of Glasgow, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375465.
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Sheikh, Qaiser Iftikhar. "Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis." Thesis, University of Sheffield, 2001. http://etheses.whiterose.ac.uk/10258/.
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Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.
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Kolb, Roman [Verfasser]. "beta-Sultams as Tools for the Study of Enzyme Function / Roman Kolb." München : Verlag Dr. Hut, 2014. http://d-nb.info/1058284762/34.
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Pretz, Monika Gyöngy. "Thermophilic P-loop transport ATPases enzyme function and energetics at high temperature /." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2006. http://irs.ub.rug.nl/ppn/299141012.
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Snow, Juliette Elizabeth. "Investigation of the function of disproportionating enzyme in potato (Solanum tuberosum L.)." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/11421.
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In this study potato plant with lowered D-enzyme activity were investigated. It was found previously that lack of D-enzyme resulted in a reduction in the tuber yield of these plants and a delay in tuber sprouting. In this study the reduction in tuber yield was characterised further. It was found that tuber fresh weight per plant was reduced but percentage dry matter and starch content were unaltered. In addition, the extent of the reduction in fresh weight tuber yield was dependent on irradiance. Lack of D-enzyme also resulted in a delay in sugar accumulation in tuber during prolonged storage. No differences were detected in the rates of starch synthesis and starch turnover in tubers with lowered D-enzyme activity compared to controls. One reason for this could have been that the labelling experiments employed to investigate tuber metabolism were not sensitive enough to detect small differences. To address this, D-enzyme activity was lowered in transgenic potato plants which exhibit exaggerated rates of starch synthesis and turnover due to the expression of a heterologous ADPglucose pyrophosphorylase gene. It was hypothesised that differences in starch metabolism would be more likely to be resolved in tubers from these plants. Surprisingly, no appreciable differences between the starch metabolism of tubers with exaggerated rates of starch turnover and lowered D-enzyme activity and control tubers were detected. However, the level of D-enzyme activity in these tubers was 14% of wild type and this could have been high enough for the <i>in vivo</i> role(s) of the enzyme to be fulfilled. Rates of starch breakdown in leaf tissue lacking D-enzyme were slightly reduced compared to controls during darkness. Lack of D-enzyme did not affect rates of starch synthesis during the light. These results are consistent with those from the D-enzyme mutant of <i>A. thaliana</i> and suggest that D-enzyme may have a role in potato leaf starch degradation during darkness and that this could in turn influence tuber growth.
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Ma, Huan. "Aldolases for Enzymatic Carboligation : Directed Evolution and Enzyme Structure-Function Relationship Studies." Doctoral thesis, Uppsala universitet, Biokemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-266902.
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The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties. In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.
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Rood, Jennifer E. (Jennifer Evelyn). "Structure and function of the human Poly(ADP-ribose) polymerase enzyme family." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81033.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>The poly(ADP-ribose) polymerase (PARP) family of enzymes in humans is comprised of 17 proteins. PARP-1, the first member of the family, synthesizes a large, complex post-translational modification, poly(ADP-ribose). While PARP-1 and some other PARPs have been extensively functionally characterized, the enzymatic and cellular functions of many PARPs are unknown. This thesis presents work that seeks to characterize the enzymatic functions of the PARP family. First, experimental demonstration of the automodification capacity of each PARP is presented. We find that PARP enzymatic activity largely conforms to bioinformatic predictions of PARP activity. Then, we seek to provide a structural rationale for these enzymatic capabilities based on the analysis of extant and modeled crystal structures of each PARP. We present a structural hypothesis for catalytic differences among PARPs. Finally, we examine methods for the identification of cellular targets of PARP activity and functional interaction partners of PARPs. Together, these elements of PARP characterization will aid in the discovery of physiologically relevant targets and a mechanistic understanding of PARP enzymatic activity in the cellular context.<br>by Jennifer E. Rood.<br>Ph.D.
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Ylianttila, M. (Mari). "Structure-function studies of the peroxisomal multifunctional enzyme type 2 (MFE-2)." Doctoral thesis, University of Oulu, 2005. http://urn.fi/urn:isbn:9514278968.
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Abstract
Multifunctional enzyme type 2 (MFE-2) catalyses the second and the third reactions in the eukaryotic peroxisomal β-oxidation cycle, which degrades fatty acids by removing a two-carbon unit per each cycle. In addition to the 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase activities, mammalian MFE-2 has also a sterol carrier protein type 2-like (SCP-2L) domain. In contrast, yeast MFE-2 has two (3R)-hydroxyacyl-CoA dehydrogenases, one 2-enoyl-CoA hydratase 2 and no SCP-2L domain.
The physiological roles of yeast (3R)-hydroxyacyl-CoA dehydrogenases (A and B) were tested by inactivating them in turn by site-directed mutagenesis and testing the complementation of Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2) with mutated variants of Sc MFE-2. Growth rates were lower for fox-2 cells expressing only a single functional domain than for those expressing the Sc MFE-2. Kinetic studies with purified Candida tropicalis MFE-2 and its mutated variants show that dehydrogenase A catalyzes the reaction more efficiently with the medium- and long-chain substrates than dehydrogenase B, which in turn is the only one active with the short chain fatty acids.
The structural basis of the substrate specificity difference of these two dehydrogenases was solved by X-ray crystallography together with docking studies. Protein engineering was used to produce a stabile, homogenous recombinant protein of C. tropicalis dehydrogenases in one polypeptide. The heterodimeric structure contains the typical fold of the short-chain alcohol dehydrogenase/reductase (SDR) family. Docking studies suggest that dehydrogenase A binds medium chain-length substrates as bended, whereas short chain substrates are dislocated, because they do not reach the hydrophobic contacts needed for anchoring the substrate to the active site, but are instead attracted by L44. Dehydrogenase B has a more shallow binding pocket and thus locates the short chain-length substrates correctly for catalysis. Thus the data provide clues for structural basis of the different substrate specificities.
The molecular basis of the patient mutations of MFE-2 (DBP deficiency) was studied using the recently solved crystal structures of rat (3R)-hydroxyacyl-CoA dehydrogenase, human 2-enoyl-CoA hydratase and SCP-2L. The predicted effect of the mutations on protein structure could in several cases be explained, and these data supported the conclusion that a genotype-phenotype correlation exists for DBP deficiency.
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Martínez, Cuesta Sergio. "The chemistry and evolution of enzyme function : isomerases as a case study." Thesis, University of Cambridge, 2014. https://www.repository.cam.ac.uk/handle/1810/246994.
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The study of the evolution of proteins has been traditionally undertaken from a sequence and structural point of view. However any attempt to understand how protein function changes during evolution benefits from consistent definitions of function and robust approaches to quantitatively compare them. The function of enzymes is described as their ability to catalyse biochemical reactions according to the Enzyme Commission (EC). This dissertation explores aspects of the chemistry and evolution of a small class of enzymes catalysing geometrical and structural rearrangements between isomers, the isomerases (EC 5). A comprehensive analysis of the overall chemistry of isomerase reactions based on bond changes, reaction centres and substrates and products revealed that isomerase reactions are chemically diverse and difficult to classify using a hierarchical system. Although racemases and epimerases (EC 5.1) and cis-trans isomerases (EC 5.2) are sensibly grouped according to changes of stereochemistry, the overall chemistry of intramolecular oxidoreductases (EC 5.3), intramolecular transferases (EC 5.4) and intramolecular lyases (EC 5.5) is challenging. The subclass \other isomerases" (EC 5.99) sits apart from other subclasses and exhibits great diversity. The current classification of isomerases in six subclasses reduces to two subclasses if the type of isomerism is considered. In addition, the separation of groups of isomerases sharing similar chemistry such as oxidosqualene cyclases and pseudouridine synthases from chemically complex sub-subclasses like intramolecular transferases acting on \other groups" (EC 5.4.99) might also improve the classification. An overview of the evolution of isomerase function in superfamilies revealed three main findings. First, isomerases are more likely to evolve new functions in different EC primary classes, especially lyases (EC 4), rather than evolve to perform different isomerase reactions. Second, isomerases change their overall chemistry and conserve the structure of their substrates and products more often than conserving the chemistry and changing substrates and products. Last, the relationship between sequence and functional similarity suggests that correlations should be investigated on the basis of closely related enzymes. Although previous research assumes a one-to-one relationship between EC number and biochemical reaction, almost one-third of all known EC numbers are linked to more than one biochemical reaction. This complexity was characterised for isomerase reactions and used to develop an approach to automatically explore it across the entire EC classification. Remarkably, about 30% of the EC numbers bearing more than one reaction are linked to different types of reactions, bearing key differences in catalysed bond changes. Several recommendations to improve the description of complex biochemical reaction data in the EC classification were proposed. This dissertation explores enzymes from a functional perspective as an alternative to classical studies based on homology. This standpoint might prove useful to help to search for sequence candidates for orphan enzymes and in the design of enzymes with novel activities.
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Sikora, Aneta E. "Structure-function analysis of a multifunctional enzyme using the atomic force microscope." Thesis, University of Portsmouth, 2010. https://researchportal.port.ac.uk/portal/en/theses/structurefunction-analysis-of-a-multifunctional-enzyme-using-the-atomic-force-microscope(fdbd2065-c230-4eba-a7cd-94cbb1bb9e16).html.
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The type I R-M enzyme EcoR124I is a multifunctional, multisubunit molecular motor with the ability to self-assemble. In the presence of hydrophobic compounds, subunit disassembly has been observed leading to the possibility of using the enzyme as a nanoactuator in toxicity biosensors. A better understanding of single molecule interactions between the subunits has been investigated using atomic force microscopy (AFM), a powerful tool for measuring forces and dynamics between single molecules with a picoNewton sensitivity. AFM imaging of DNA fragments with a single recognition binding site for EcoR124I positioned in the middle or at 1/3 of the length of DNA, was used to study the assembled holoenzyme. Reproducible DNA imaging was investigated using divalent cations (Mg2+, Ca2+, Ni2+). The presence of only one EcoR124I holoenzyme bound to DNA was observed, confirming the specificity of binding. Molecular volume (Vm) measurements were used to identify subunits and complexes. The effect of ATP analogues (ATP-γ-S and AMP-pnp) on enzyme stability was also investigated. The addition of ATP, although not novel, confirmed the enzyme activity by showing the ability of the enzyme to translocate. Biotin-avidin interactions were studied using AFM force curves as a model to probe the novel HsdR-MTase system. AFM tips were functionalised using both glutaraldehyde and a PEG linker. In the former, many multiple event force curves were seen, although the final “pull-off” event yielded information on single-molecule or near single-molecule interactions: a single biotin-avidin interaction at 56 ± 13 pN was measured, with further periodic force maxima at 98 ± 15 and 161 ± 3 pN (two and three interactions, respectively). The use of a PEG linker allowed more sensitive measurements to be made, with a single biotin-avidin interaction at 47 ± 9.5 pN and, again, periodic maxima were seen at 93 ± 7 and 143 ± 4 pN. The PEG linker method allowed more single molecules interactions to be measured (ca. 70% of analysed forcedistance curves). Forces between a GST-HsdR(PrrI) motor subunit attached to an AFM tip using a PEG linker and MTase on poly-L-lysine pre-treated mica were studied using dynamic force spectroscopy (DFS). A single barrier in the energy landscape of the complex was found in the dissociation pathway (xdiss) to be located 13.5 Å from the bound state. The value kdiss for the GST-HsdR(PrrI)-MTase complex was calculated to be 0.16 s-1 and the lifetime t(0) of the GST-HsdR(PrrI)-MTase bond was found to be 6.25 s. GST – anti GST antibody interactions and HsdR – anti-GST antibody interactions suggest that forces measured between HsdR and MTase were realistic for the GSTHsdR(PrrI)-MTase complex.
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Hidestrand, Mats. "Structure and function of hepatic cytochromes P450 - implications for drug development /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-418-6/.
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Millar, Timothy Marc. "Novel aspects of the activity and function of xanthine oxidase." Thesis, University of Bath, 1999. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311326.
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Chu, Yuanyuan. "Role of the ubiquitin-editing enzyme A20 in B cell function and disease." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-151826.
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Gross, Martin. "The tryptophan residues of mitochondrial creatine kinase : roles in enzyme structure and function /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10719.
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Sliz, Piotr. "Structure, function and interactions of enzyme IIA from the phosphoenolpyruvate, lactose phosphotransferase system." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0020/NQ53658.pdf.
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Carter, Lisa, Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Mutagenesis of a Flavonol- 3-O-Glucosyltransferase and the Effect on Enzyme Function." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/349.
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Flavonoids are an important group of secondary metabolites found in plants and have a wide variety of properties. Some play a role in fl ower pigmentation, while others have antimicrobial properties. Glucosylation is an important modifi cation of fl avonoids and is mediated by glucosyltransferases. In this process, the enzyme transfers glucose from UDP-glucose to a specifi c position on the fl avonoid. Previous study from the lab characterized a glucosyltransferase from C. paradisi that is fl avonol specifi c. In this study an attempt has been made to study the structure and function of this fl avonol specifi c glucosyltransferase using site directed mutagenesis. The glutamine residue at position 87 of the Cp-3-O-GT enzyme was changed to isoleucine, the analogous residue in the 3-O-glucosyltransferase of Clitoria ternatea. Similarly, the histidine at position 154 was changed to tyrosine. We hypothesize that these mutations will change substrate specifi city. The glutamate at position 88 was changed to an aspartic acid. We hypothesize that this will change the regiospecifi city of the enzyme, as aspartic acid is the analogous residue found in some 7-O-glucosyltransferases. Finally, we introduced a double mutation with glutamine 87 becoming isoleucine and glutamate 88 becoming aspartic acid, with the hypothesis that both regiospecifi city and substrate specifi city will be changed.
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Myllykoski, M. (Matti). "Structure and function of the myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase." Doctoral thesis, Oulun yliopisto, 2013. http://urn.fi/urn:isbn:9789526201375.
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Abstract The myelin sheath is a crucial component of vertebrate nervous systems. Myelin is formed as the plasma membrane of a glial cell is wrapped around a neuronal axon. The presence of myelin enables the fast transmission of neuronal impulses, and degradation or dysfunction of myelin results in severe neurological symptoms. Molecular composition of myelin is unique, and many myelin proteins are not present elsewhere in the body. A myelin enzyme, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), is found in specific regions within the myelin sheath and is one of the most abundant proteins in the brain. Substrates for CNPase catalytic activity are formed during brain damage. CNPase also interacts with the cytoskeleton and cell membranes, and it is thought to play a role during myelin formation. Mice that lack CNPase suffer from axonal degeneration and die early. The aim of this study was to characterise CNPase structure and function. To this end, a system was first developed to produce the protein for subsequent analyses. The aim was to characterise the catalytic mechanism of CNPase by determining its three-dimensional molecular structure at different stages of the catalytic reaction. The interactions between CNPase and other molecules related to its function would also be characterised. Finally, the structure of the full-length protein would be used to understand of the function of the uncharacterised N-terminal domain. Using X-ray crystallography, the structure of the CNPase catalytic domain was determined in the presence of substrate and product molecules. These data, complemented with analyses of mutationally inactivated enzyme variants, were used to examine the catalytic reaction at the molecular level. The catalytic domain structure was compared to homologous enzymes from diverse organisms. The interaction between CNPase and the calcium-sensing protein calmodulin was characterised. The solution structure of full-length CNPase was determined using small-angle X-ray scattering, and protein sequence databases were utilised to determine CNPase conservation during animal evolution. The results provide novel information on the catalytic activity and overall function of CNPase. Further studies will be necessary to determine its specific role, but it is increasingly clear that CNPase can perform multiple important tasks within the nervous system<br>Tiivistelmä Myeliinituppi on tärkeä osa selkärankaisten hermostoa. Myeliiniä muodostuu, kun gliasolun solukalvo kiertyy hermosolun aksonin ympärille. Myeliini mahdollistaa hermoimpulssien nopean välityksen, ja sen tuhoutuminen ja vajaatoiminta aiheuttavat vakavia neurologisia oireita. Myeliinin molekyylikoostumus on ainutlaatuinen, ja monet myeliiniproteiineista eivät esiinny muualla elimistössä. Myeliinissä esiintyvää entsyymiä, 2′,3′-syklisten nukleotidien 3′-fosfodiesteraasia (CNPaasi), esiintyy runsaasti tietyillä myeliinialueilla, ja se on yksi aivojen runsaslukuisimmista proteiineista. Substraatteja CNPaasin katalyyttiselle aktiivisuudelle muodostuu aivovaurion aikana. CNPaasi on myös vuorovaikutuksessa solun tukirangan ja solukalvon kanssa, ja sen uskotaan vaikuttavan myeliinin muodostumiseen. Hiiret, joilta puuttuu CNPaasi, kärsivät aksonien rappeumista ja kuolevat ennenaikaisesti. Tämän tutkimuksen tavoite oli karakterisoida CNPaasin rakennetta ja toimintaa. Tätä tarkoitusta varten ensin kehitettiin menetelmä analysoitavan proteiinin tuottamiseksi. Tavoitteena oli karakterisoida CNPaasin katalyyttinen mekanismi määrittämällä sen kolmiulotteinen molekyylirakenne katalyysireaktion eri vaiheissa. Myös CNPaasin vuorovaikutuksia sen toimintaan liittyvien molekyylien kanssa tutkittiin. Lopuksi kokopitkän proteiinin rakenteen avulla selvitettiin karakterisoimattoman aminoterminaalisen alayksikön toimintaa. CNPaasin katalyyttisen alayksikön rakenne määritettiin käyttäen röntgenkristallografiaa substraatti- ja tuotemolekyylien läsnäollessa. Rakennetta, täydennettynä mutaatioilla inaktivoitujen entsyymimuunnosten analyysillä, käytettiin katalyyttisen reaktion molekyylitason karakterisointiin. Katalyyttisen alayksikön rakennetta verrattiin eri organismeissa esiintyviin homologisiin entsyymeihin. CNPaasin ja kalsiumia sitovan kalmoduliinin vuorovaikutusta karakterisoitiin. Kokopitkän CNPaasin liuosrakenne selvitettiin pienkulmaröntgensironnan avulla, ja CNPaasin sekvenssin säilymistä eläinten evoluution aikana tarkasteltiin proteiinisekvenssitietokantoja käyttämällä. Tulokset antavat uutta tietoa CNPaasin katalyyttisestä aktiivisuudesta ja tämän arvoituksellisen entsyymin toiminnasta. Jatkotutkimukset ovat tarpeen sen täsmällisen roolin selvittämiseksi, mutta on kasvavassa määrin selvää, että CNPaasi pystyy suorittamaan useita tärkeitä tehtäviä hermostossa
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Haapalainen, A. (Antti). "Structure-function studies of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2)." Doctoral thesis, University of Oulu, 2002. http://urn.fi/urn:isbn:9514268385.
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Abstract
Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2E)-enoyl-CoA hydratase-2 and (3R)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2).
The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved.
The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer.
The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes.
The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid in vivo was tested with Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2). Subsequently, in vitro characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.
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Just, Sissy [Verfasser]. "T cell specific function of the deubiquitinating enzyme A20 in murine listeriosis / Sissy Just." Magdeburg : Universitätsbibliothek, 2017. http://d-nb.info/1128726459/34.
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McIntosh, Cecilia A. "Structure and Function of Flavonoid Glucosyltransferases: Using a Specific Grapefruit Enzyme as a Model." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/355.
Full textAbstract:
Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.
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McIntosh, Cecilia A. "Structure and Function of Flavonoid Glucosyltransferases: Using a specific Grapefruit Enzyme as a Model." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/369.
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