Matching Items (14)

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Incorporation, Expression, and Retained Enzymatic Activity of Six Tryptophan Analogues in Dihydrofolate Reductase at Positions 30 and 47

Description

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site amino acid replacements. The two positions 30 and 47 within DHFR were studied to discover the rate at which these larger tryptophan analogues may be incorporated. Additionally, it was to be determined how much activity the mutated DHFR’s could retain when compared to their wild type counterpart. Through a review of literature, it was shown that previous studies have illustrated successful incorporation and toleration of unnatural amino acids.
Each of the six analogues A through F were relatively efficiently incorporated into the enzyme and well tolerated. Each maintained at least a third of their catalytic activity, measured through the consumption of β-nicotinamide adenine dinucleotide phosphate. Primarily, derivatives B, C, and D were able to retain the highest amount of activity in each position; B and D were the most tolerated in positions 30 and 47 with respective values of 68 ± 6.1 and 80 ± 12. The findings in this study illustrate that single tryptophan derivatives are able to be incorporated into Escherichia coli DHFR while still allowing the maintenance of a significant portion of its enzymatic activity.

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  • 2015-05

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Exploration of Enzymatic Efficiency in Double-Stranded DNA by Uracil-DNA Glycosylase and Optimization of Glycosylation Reaction of DNA Precursor

Description

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.

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Date Created
  • 2016-05

Bio-Inspired Cementation of Soil Using Plant Enzyme

Description

This dissertation investigates the potential for enzyme induced carbonate cementation as an alternative to Portland cement for creating building material from sand aggregate. We create a solution of urease enzyme,

This dissertation investigates the potential for enzyme induced carbonate cementation as an alternative to Portland cement for creating building material from sand aggregate. We create a solution of urease enzyme, calcium chloride (CaCl2), and urea in water and added sand. The urease catalyzes the synthesis of carbonate from urea, and the carbonate then bonds with a dissociated calcium ion and precipitates from the solution as calcium carbonate (CaCO3). This precipitate can form small crystal bridges at contacts between sand grains that lock the sand grains in place. Using enzyme induced carbonate precipitation we created a cemented sand sample with a maximum compressive strength of 319 kPa and an elastic modulus of approximately 10 MPa. Images from the SEM showed that a major failure mechanism in the cemented samples was the delamination of the CaCO3 from the sand grains. We observed that CaCO3 cementation did not when solutions with high concentrations of CaCl2 and urea were used.

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  • 2014-05

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Metal Replacement Studies in Bacillus subtilis Quercetinase

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Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to

Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to the corresponding depside and carbon monoxide. The first quercetinase was characterized from a species of Aspergillus genus, and was found to contain one Cu2+ per subunit. For many years, it was thought that the B. subtilis quercetinase contained two Fe2+ ions per subunit; however, it has since been discovered that Mn2+ is a much more likely cofactor. Studies of overexpressed bacterial enzyme in E. coli indicated that this enzyme may be active with other metal ions (e.g. Co2+); however, the production of enzyme with full metal incorporation has only been possible with Mn2+. This study explores the notion that metal manipulation after translation, by partially unfolding the enzyme, chelating the metal ions, and then refolding the protein in the presence of an excess of divalent metal ions, could generate enzyme with full metal occupancy. The protocols presented here included testing for activity after incubating purified quercetinase with EDTA, DDTC, imidazole and GndHCl. It was found that the metal chelators had little to no effect on quercetinase activity. Imidazole did appear to inhibit the enzyme at concentrations in the millimolar range. In addition, the quercetinase was denatured in GndHCl at concentrations above 1 M. Recovering an active enzyme after partial or complete unfolding proved difficult, if not impossible.

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Date Created
  • 2014-05

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Universal Biochemistry Within and Across Biological Domains and Levels of Organization on Earth

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Universal biology is an important astrobiological concept, specifically for the search for life beyond Earth. Over 1.2 million species have been identified on Earth, yet all life partakes in certain

Universal biology is an important astrobiological concept, specifically for the search for life beyond Earth. Over 1.2 million species have been identified on Earth, yet all life partakes in certain processes, such as homeostasis and replication. Furthermore, several aspects of biochemistry on Earth are thought to be universal, such as the use of organic macromolecules like proteins and nucleic acids. The presence of many biochemical features in empirical data, however, has never been thoroughly investigated. Moreover, the ability to generalize universal features of Earth biology to other worlds suffers from the epistemic problem of induction. Systems biology approaches offer means to quantify abstract patterns in living systems which can more readily be extended beyond Earth’s familiar planetary context. In particular, scaling laws, which characterize how a system responds to changes in size, have met with prior success in investigating universal biology.

This thesis rigorously tests the hypothesis that biochemistry is universal across life on Earth. The study collects enzyme data for annotated archaeal, bacterial, and eukaryotic genomes, in addition to metagenomes. This approach allows one to quantitatively define a biochemical system and sample across known biochemical diversity, while simultaneously exploring enzyme class scaling at both the level of both individual organisms and ecosystems. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Joint Genome Institute’s Integrated Microbial Genomes and Microbiomes (JGI IMG/M) database, this thesis performs the largest comparative analysis of microbial enzyme content and biochemistry to date. In doing so, this thesis quantitatively explores the distribution of enzyme classes on Earth and adds constraints to notions of universal biochemistry on Earth.

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Date Created
  • 2020

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Development of an artificial genetic system capable of Darwinian evolution

Description

The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of

The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.

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Date Created
  • 2013

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From plasma peptide to phenotype: the emerging role of quiescin sulfhydryl oxidase 1 in tumor cell biology

Description

Cancer is a disease that affects millions of people worldwide each year. The metastatic progression of cancer is the number one reason for cancer related deaths. Cancer preventions rely on

Cancer is a disease that affects millions of people worldwide each year. The metastatic progression of cancer is the number one reason for cancer related deaths. Cancer preventions rely on the early identification of tumor cells as well as a detailed understanding of cancer as a whole. Identifying proteins specific to tumor cells provide an opportunity to develop noninvasive clinical tests and further our understanding of tumor biology. Using liquid chromatography-mass spectrometry (LC-MS/MS) a short peptide was identified in pancreatic cancer patient plasma that was not found in normal samples, and mapped back to QSOX1 protein. Immunohistochemistry was performed probing for QSOX1 in tumor tissue and discovered that QSOX1 is highly over-expressed in pancreatic and breast tumors. QSOX1 is a FAD-dependent sulfhydryl oxidase that is extremely efficient at forming disulfide bonds in nascent proteins. While the enzymology of QSOX1 has been well studied, the tumor biology of QSOX1 has not been studied. To begin to determine the advantage that QSOX1 over-expression provides to tumors, short hairpin RNA (shRNA) were used to reduce the expression of QSOX1 in human tumor cell lines. Following the loss of QSOX1 growth rate, apoptosis, cell cycle and invasive potential were compared between tumor cells transduced with shQSOX1 and control tumor cells. Knock-down of QSOX1 protein suppressed tumor cell growth but had no effect on apoptosis and cell cycle regulation. However, shQSOX1 dramatically inhibited the abilities of both pancreatic and breast tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, shQSOX1-transduced tumor cells secreted MMP-2 and -9 that were less active than MMP-2 and -9 from control cells. Taken together, these results suggest that the mechanism of QSOX1-mediated tumor cell invasion is through the post-translational activation of MMPs. This dissertation represents the first in depth study of the role that QSOX1 plays in tumor cell biology.

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Date Created
  • 2012

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Exploring biosynthetic pathways for aromatic ester production

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Synthetic biology and metabolic engineering has aided the production of chemicals using renewable resources, thus offering a solution to our dependence on the dwindling petroleum resources. While a major portion

Synthetic biology and metabolic engineering has aided the production of chemicals using renewable resources, thus offering a solution to our dependence on the dwindling petroleum resources. While a major portion of petroleum resources go towards production of fuels, a significant fraction also goes towards production of specialty chemicals. There has been a growing interest in recent years in commercializing bio-based production of such high value compounds. In this thesis the biosynthesis of aromatic esters has been explored, which have typical application as flavor and fragrance additive to food, drinks and cosmetics. Recent progress in pathway engineering has led to the construction of several aromatic alcohol producing pathways, the likes of which can be utilized to engineer aromatic ester biosynthesis by addition of a suitable enzyme from the acyltransferase class. Enzyme selection and screening done in this work has identified chloramphenicol O-acetyltransferase enzyme(CAT) as a potential candidate to complete the biosynthetic pathways for each of 2-phenethyl acetate, benzyl acetate, phenyl acetate and acetyl salicylate. In the end, E. coli strains capable of producing up to 60 mg/L 2-phenethyl acetate directly from glucose were successfully constructed by co-expressing CAT in a previously engineered 2-phenylethanol producing host.

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Date Created
  • 2016

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Small molecule inhibition of quiescin sulfhydryl oxidase 1 (QSOX1), a dynamic pro-tumorigenic regulator of the extracellular matrix

Description

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.

The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.

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Date Created
  • 2015

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DNA nanostructures as programmable biomolecular scaffolds for enzymatic systems

Description

Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation

Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation positioning, substrate channeling, compartmentalization, and more.

DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.

This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.

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Date Created
  • 2016