Browsing by Subject "Enzymes"

Now showing 1 - 3 of 3
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    Characterization and evolution of artificial RNA ligases
    (2015-06) Morelli, Aleardo
    Enzymes enable biocatalysis with minimal by-products, high regio- and enantioselectivity, and can operate under mild conditions. These properties facilitate numerous applications of enzymes in both industry and research. Great progress has been made in protein engineering to modify properties such as stability and catalytic activity of an enzyme to suit specific processes. On the contrary, the generation of artificial enzymes de novo is still challenging, and only few examples have been reported. The study and characterization of artificial enzymes will not only expand our knowledge of protein chemistry and catalysis, but ultimately improve our ability to generate novel biocatalysts and engineer those found in nature. My thesis focused on the characterization of an artificial RNA ligase previously selected from a library of polypeptide variants based on a non-catalytic protein scaffold. The selection employed mRNA display, a technique to isolate de novo enzymes in vitro from large libraries of 1013 protein variants. The artificial RNA ligase catalyzes the formation of a phosphodiester bond between two RNA substrates by joining a 5'-triphospate to a 3'-hydroxyl, with the release of pyrophosphate. This activity has not been observed in nature. An initial selection carried out at 23°C yielded variants that were poorly suitable for biochemical and biophysical characterization due do their low solubility and poor folding. We hence focused our studies on a particular improved ligase variant called ligase 10C, isolated from a subsequent selection performed at 65°C. Here we report the structural and biochemical characterization of ligase 10C. We solved the three-dimensional structure of this enzyme by NMR. Unexpectedly, the original structure of the parent scaffold used for building the original library was abandoned. The enzyme instead adopted a novel dynamic fold, not previously observed in nature. The structure was stabilized by metal coordination, yet lacked secondary structural motifs entirely. We also compared the catalytic and thermodynamic properties of ligase 10C to enzyme variants previously selected at lower temperature (23°C). Ligase 10C displayed a remarkable increase in melting temperature of 35°C compared to its mesophilic counterpart. In addition, its activity at 23°C was about 10-fold higher compared to the mesophilic variants. This work was the first mRNA display selection for catalytic activity at high temperature, and further highlighted the capacity of the technique to select for proteins with rare properties. To facilitate detailed mechanistic studies of this unnatural enzyme, a crystal structure would be essential. Unfortunately, ligase 10C did not form crystals likely due to its highly dynamic regions. With the goal of identifying a truncated less flexible version of the enzyme that would be more suited for crystallization, we generated a library of random deletion variants of ligase 10C and performed an mRNA display selection to identify shorter active variants. Finally, we describe the attempted selection of an enzyme for the same RNA ligation reaction from a completely random polypeptide library. The long-term goal of the overarching project in the Seelig lab is to elucidate and compare the structure and mechanism of enzymes generated from different starting points, yet catalyzing the same reaction, to obtain insights into potential evolutionary pathways. In summary, our work revealed the unusual structural and biophysical properties of the artificial ligase 10C, and thereby demonstrated the power and flexibility of mRNA display as a technique for the selection of de novo enzymes.
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    Development of computational tools for modeling the biotransport of small organic molecules into the active site of broad-substrate specificity enzymes
    (2019-07) Escalante, Diego
    In this dissertation research, two new computational tools were developed to model the biotransport of small organic molecules into the active site of broad-substrate specificity (BSS) enzymes. The biological organism selected to develop, test and validate these tools were Rieske non-heme iron dioxygenases. Members of this family of enzymes are known to have biocatalytic activity on more than three hundred different substrates. The large diversity of substrates that can be acted upon makes these enzymes very attractive in biotechnological processes such as bioremediation. In addition, the highly specific chirality of the products obtained makes these enzymes attractive for the potential synthesis of pharmaceutical precursors. Currently, the most common way to identify new substrates requires formulating an educated guess followed by the arduous task of testing each possible compound individually. This slows down the pace at which new industrial processes can be formulated or current ones further developed. The tools presented in this research provide fundamental and practical scientific contributions. For the basic science studies of my dissertation, an all-atom and, a coarse-grained (CG) model of Rieske non-heme iron dioxygenases were used to investigate the factors that affect the biotransport of small organic molecules into their active sites. From the all-atom model I discovered a gating mechanism that allows aromatic substrates into the active site and blocks other compounds. The key to these gates are T-stacked pi-pi interactions between hydrophobic amino acids and the aromatic substrates. On the other hand, from the CG model I discovered that the shape of tunnel modulates the hydrophobicity level of the surface. As the tunnels become more concave, the hydrophobicity increases causing the formation of a water exclusion zone which increases the diffusivity of aromatic substrates. The CG models also revealed that convex tunnels prevent the adhesion of hydrophobic substrates to the tunnel walls; providing a possible explanation for the evolution of bottlenecks at the entrance of Rieske active sites. For the practical contributions of my dissertation, I developed two new computational tools for the prediction of Rieske substrates. The first tool is an all-atom algorithm that models the stochastic roto-translational movement of small organic molecules along the Rieske enzyme tunnels. This algorithm has a 92% prediction accuracy of Rieske substrates. In addition, it is capable of elucidating the location of high-energy barriers along the tunnel, allowing the formulation of possible protein engineering sites. The second tool is a CG non dimensional model of the Rieske enzyme tunnels. This algorithm has a 90% prediction accuracy of Rieske substrates. The processing time of 1ms/substrate combined with its high accuracy allows for the high-throughput screening of possible Rieske substrates.
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    Effects of multiple exogenous enzyme products on in vitro fermentation by rumen microbes in batch and continuous culture fermentation
    (2019-08) Dado, Trent
    Research has demonstrated that supplementing exogenous enzymes to ruminants has potential to improve feed digestion and animal performance. Enzyme products with fibrolytic, proteolytic and amylolytic activities and diets with diverse composition have been used to test enzyme efficacy. Responses to these conditions have been variable. A series of in vitro experiments were designed to 1) screen enzyme products in ruminal batch culture to determine effects on digestibility and gas production and 2) further examine effective enzymes in dual-flow continuous culture to determine their effect on microbial fermentation. In Exp. 1, seven treatments, including a multi-enzyme blend (MEB), ferulic acid esterase (FAE), protease (PRO), α-amylase (AAM), β-glucanase (BGL), xylanase (XYL) or control (CON), were added to each of 3 diets with forage:concentrate ratios of 50:50 (Diet 1), 30:70 (Diet 2) or 10:90 (Diet 3), at 6 dosage rates (0, 125, 250, 500, 1000 and 10,000 mg of enzyme/kg of diet DM) in batch culture. Dose was removed from analysis and all doses were combined and examined together. In vitro total dry matter digestibility (IVTDMD) was greater (P < 0.05) for BGL compared with all other treatments in Diet 1 and Diet 2. In Diet 3, BGL was greater (P < 0.05) than AAM and CON. Control and XYL had greater (P < 0.05) total gas production than MEB, FAE, and AAM. Rate of gas production (mL/h/g of DM) was also affected (P < 0.05) by diet and enzyme for the first 24 h of fermentation with CON having the fastest rate at 3 h, generally. Overall, BGL increased digestibility without generating as much gas as CON. In Exp. 2, BGL and PRO from Exp. 1, a cellulase (CEL) preparation and control (CON) were examined in eight dual-flow continuous culture over 3 periods. The diet was a 40:60 (forage:concentrate) and enzyme was supplied at 1000 mg/kg of diet DM. Digestibility of apparent DM and OM tended to be greater (P < 0.10) for CON compared with CEL. Volatile fatty acid (VFA) concentration and nitrogen metabolism were not affected (P > 0.05) by enzyme treatment. In summary, the increase in DM and OM digestibility found with BGL and other effects of enzymes on fermentation in batch culture were not observed in dual-flow continuous culture.