Browsing by Subject "Enzymes"
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Item Biochemical studies of the enzymes involved in deoxysugar D-forosamine biosynthesis(2004) Hong, Lin, 1976-; Liu, Hung-wen, 1952-Deoxysugars are indispensable structural components of many biologically active natural products and are essential for many significant cellular processes. In this work, the biosynthetic pathway of TDP-forosamine, which is a 2,3,4,6-tetradeoxy sugar component of the potent, and environmentally benign insecticide spinosyn, has been established without ambiguity. Five genes, spnO, spnN, spnQ, spnR, and spnS have been cloned from Saccharopolyspora spinosa chromosomal DNA and expressed in E. coli. The encoded proteins have been purified and characterized by in vitro assays. The products of four reactions have been isolated and characterized with MS and/or NMR. SpnO was demonstrated to be a 2,3-dehydratase, while SpnN was shown to be a 3- ketoreductase, catalyzing equatorial hydroxyl formation at C-3. Studies of the combined action of SpnO and SpnN reinforce the previous finding that C-2 deoxygenation is accomplished through a -elimination/reduction type mechanism. SpnQ is a homologue of the well-characterized CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) which in combination with CDP-6-deoxy-4-keto-3,4-glucoseen reductase (E3) catalyzes the C-O bond cleavage at C-3 in the biosynthesis of CDP-ascarylose in Yersinia pseudotuberculosis. In the biosynthesis of TDP-forosamine, SpnQ was shown to catalyze an analogous C-3 deoxygenation but instead using ferredoxin/ferredoxin reductase pair as the electron transfer media. Additionally, in the absence of ferredoxin/ferredoxin reductase to allow C-3 deoxygenation, and in the presence of Lglutamate as an amino donor, SpnQ was shown to act as a 4-aminotransferase, converting TDP-2,6-dideoxy-4-keto-D-glucose to TDP-4-amino-2,4,6-trideoxy-D-glucose. Due to the instability of 2-deoxysugars, an analog of TDP-4-amino-2,3,4,6-tetradeoxy-glucose was used to characterize the late steps in the pathway. SpnR was shown to be a 4- aminotransferase which, interestingly, can recognize both the SpnQ product and the SpnN product as substrates. SpnS was shown to be a 4-amino-N,N-dimethyltransferase. Mechanistic investigation revealed that both N-methyl groups of TDP-forosamine are installed by SpnS in a step-wise manner via a monomethylated intermediate. Together, studies described in this thesis have extensively enriched our knowledge of the enzymes involved in deoxysugar biosynthesis. The newly discovered substrate flexibility of TDPforosamine pathway enzymes will also be very useful in generating engineered sugar biosynthetic pathway for use in generating novel natural products.Item The bioelectrochemistry of enzymes and their cofactors at carbon nanotube and nitrogen-doped carbon nanotube electrodes(2014-05) Goran, Jacob Michael; Stevenson, Keith J.; Crooks, Richard M; Kirisits, Mary J; Keatinge-Clay, Adrian T; Brodbelt, Jennifer SThis dissertation explores the electrochemical behavior of enzymes and their cofactors at carbon nanotube (CNT) and nitrogen-doped carbon nanotube (N-CNT) electrodes. Two common types of oxidoreductases are considered: flavin adenine dinucleotide (FAD)-dependent oxidases and nicotinamide adenine dinucleotide-dependent (NAD⁺)-dehydrogenases. Chapter 1 presents the oxygen reduction reaction (ORR) at N-CNT electrodes as a way to electrochemically measure enzymatic turnover at the electrode surface. The unique peroxide pathway at N-CNT electrodes, which catalytically disproportionates hydrogen peroxide (H₂O₂) back into oxygen, provides an increased ORR current directly proportional to the rate of enzymatic turnover for H₂O₂ producing enzymes, even in an oxygen saturated solution. Biosensing of L-lactate using the increased ORR current is demonstrated using L-lactate oxidase. Chapter 2 explores the surface bound electrochemical signal of FAD when FAD-dependent enzyme or free FAD is allowed to spontaneously adsorb onto the CNT/N-CNT surface. Specifically, the origin of the enzymatically generated FAD signal and the rate constant of the electron transfer are elucidated. Chapter 3 continues the discussion of the cofactor FAD by demonstrating its use as an informative surface specific redox probe for graphitic carbon surfaces. Primarily, FAD can be used to determine the electroactive surface area and the relative hydrophobicity/hydrophilicity of graphitic surfaces. Chapter 4 changes gears to NAD⁺-dependent dehydrogenases by investigating the electrocatalytic oxidation of NADH at N-CNTs in comparison with conventional carbon electrodes or nondoped CNTs. Biosensing of glucose through the oxidation of NADH is demonstrated using glucose dehydrogenase adsorbed onto the N-CNT surface. Chapter 5 continues the discussion of NAD⁺-dependent dehydrogenases by addressing the reaction kinetics of NADH oxidation at N-CNTs as a tool to measure the enzymatic reduction of NAD⁺.Item CDNA Cloning and Characterization of Enzymes That Synthesize Bile Acids, Vitamin D and Waxes(2006-05-15) Cheng, Jeffrey Binyan; Russell, DavidCountless enzymes are required for the synthesis of the diverse array of lipids found in nature. The identification and characterization of five different lipid metabolizing enzymes are reported here. The 3beta -hydroxy-delta 5-C27-steroid oxidoreductase (C27 3beta-HSD) enzyme catalyzes a step in bile acid synthesis. Subjects with mutations in the encoding gene fail to synthesize bile acids and develop liver disease. Fifteen patients were screened and twelve different mutations were identified in the C27 3beta -HSD gene. Vitamin D is required for normal bone metabolism and maintenance of serum calcium levels. The conversion of vitamin D into an active ligand requires 25-hydroxylation. I report here the identification by expression cloning of a cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. A patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency was identified. Molecular analysis of this individual revealed homozygosity for a transition mutation in the CYP2R1 gene causing the substitution of a proline for a leucine in the protein and eliminating vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. The reduction of fatty acids to fatty alcohols, by a fatty acyl-CoA reductase enzyme, is required for the synthesis of wax monoesters and ether lipids. Using a bioinformatics approach, the first two mammalian fatty acyl-CoA reductase genes (FAR1 and FAR2) were identified. The two mouse FAR enzymes, which share 57% sequence identity at the amino acid level, have differing substrate specificities and tissue distributions implying unique physiological roles for each. Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. Here, I report the identification by expression cloning of a wax synthase gene. Co-expression of cDNAs specifying FAR1 and wax synthase led to the synthesis of wax monoesters. The data suggests that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.Item Changes in DNA-dependent RNA polymerase I during ecdysterone induced development of the silkmoth, A. luna(Texas Tech University, 1991-08) Mattes, Carol EMuch of modern biological research focuses on the molecular mechanisms of signal transmission via the membrane or through intracellular compartments which finally influence the pattern and/or intensity of gene expression in the cell nucleus. It is surmised that cis and trans acting factors affect the formation of the preinitiation complexes by the RNA polymerase enzymes. Three distinct and structurally complex classes of RNA polymerases are involved in this process of eukaryotic gene expression called transcription, RNA polymerases I and ill transcribe rRNAs and small RNAs respectively while RNA polymerase II transcribes mRNA. rRNA synthesis is intimately linked to cell cycle and growth and/or differentiation in response to growth factors and hormones. One example is the tissues of diapausing silkmoths which respond to ecdysterone, the steroid hormone of arthropods, by enhanced RNA synthesis and RNA polymerase activities. The RNA polymerase I activity of the wing epidermis increases nine-fold within 24 hours. The mechanisms for such enhancement, which is seen in almost all instances of stimulated growth, remains unresolved. An immunological approach to measure the amount of the enzyme based on subunit specific antibodies has been developed. For this purpose, three classes of enzymes were purified from the silkglands. Monoclonal antibodies specific for three subunits of RNA polymerase I (135, 55, and 22 kDa) were produced and utilized to develop a highly reliable, reproducible, and sensitive immunoassay involving antigen capture by immobilized antibodies. Quantitation of the enzyme in ecdysone-stimulated wing epidermal extracts showed increases of only 3-fold in the RNA polymerase I content during the first 8 hours of stimulation and a 4.5-fold increase during the 24-hour time period compared to the nine-fold increase in activity at 24 hours. The covalent modification of RNA polymerase I by phosphorylation as a mechanism to explain the difference has been pursued. Several subunits are phosphorylated at serine and threonine residues which are susceptible to removal by alkaline phosphatase. However, the correlation between increased activity and modification by phosphorylation is equivocal.Item Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications(2009-12) Serrano, Hector, doctor of pharmacy; Whitman, Christian P.Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily.Item Development of enzyme-based sensor arrays(2001-08) Curey, Theodore Edward; Shear, Jason B.Item Effects of multiple stabilized enzymes on performance of steers during backgrounding and subsequent high concentrate feeding(Texas Tech University, 1999-05) Clyburn, Bradley S.The use of enzyme products for application in the animal feed industry has magnified over the past ten years, and the fermentation industry is quickly expanding to meet consumer and industry needs (Walsh and Moore, 1995). One reason for the rapid expansion in this area is consumer demand for a more naturally produced product that minimizes the amount of antibiotics incorporated into livestock feeds. Recent production trials have increased consumer awareness and concern about the use of antibiotics and growth stimulants in the livestock feed industry and therefore increased interest in evaluating the effects of direct-fed microbials (DFM) on animal performance (Yoon and Stern, 1996). Previous research concerning DFM, has illustrated an improvement in digestibility of feedstuffs and an increase in performance of growing livestock. Wiedmeier et al. (1987) reported an increase in DMD and crude protein digestibility when Aspergillus oryzae and a combination of A. oryzae and yeast cultures were fed to Holstein cattle (Bos Taurus). Similar results were illustrated by Beharka et al. (1991) when growing calves were supplemented with A. oryzae. Gomez-Alarcon et al. (1990) reported a 29% increase in digestibility with the use of A. oryzae. Reports have also shown that the rate, but not extent of fiber digestion may be Increased by fungal supplements (Caton et al., 1993). Rumen fluid analysis of calves supplemented with A. oryzae showed higher total volatile fatty acids (VFA) when compared to calves that were not supplemented with A. oryzae. In addition studies have indicated that DFM have the ability to modulate rumen fennentation (Williams and Newbold, 1990). Multiple Stabilized Enzymes is a commercial feed additive that is designed to increase the digestibility of feedstuffs and improve feed efficiency in all classes of livestock. The MSE feed additive is a molasses-based product that contains multiple stabilized enzymes, four strains of bacteria (three Lactobacillus acidophilus and one strain of Bacillus subtilis), three strains of yeast (Saccharomyces cerevisiae); three strains of fungi (two oi Aspergillus oryzae and one of Aspergillus niger) and vitamins and minerals that are crucial to livestock. The potential benefits from feeding MSE to receiving and finishing beef cattle are to improve average daily gain (ADG), feed efficiency (FE), carcass weight, carcass quality; to enhance immunity; and to increase milk yields by dairy cattle. The objectives of this study were to compare the use of ionophoresantibiotics to MSE and determine their effects on ADG, FE, carcass yield, quality grades, liver abscesses, immunity and dry matter digestibility (DMD).Item Electron paramagnetic resonance studies of the oxygenase mechanism of ribulose 1,5-bisphosphate carboxylase(Texas Tech University, 1984-05) Mattes, Carol ENot availableItem Enzymatic degradation of cotton used for spill absorption(Texas Tech University, 2004-12) Kota, BinduOil spills in marine environments produce observable impacts on the ecosystems and require immediate responses. Methods to minimize the impacts involve clean-up and collection of oil by in situ burning, biodegradation, dispersants, booms, skimmers, and sorbent materials. Cellulose-based organic sorbent materials have the advantages of selective removal of oil over water, biodegradability, relatively low cost, and limited impact on the environment. Cotton and wool fibers could replace synthetic materials, such as polypropylene, as the sorbents of choice in oil spill removal. Cotton and wool fibers biodegrade, preferentially adsorb oil over water due to the natural wax coat on their surfaces, and can be easily disposed. Cellulose sorbents are also used in cleaning and maintenance of equipment in laboratories. Generally, cheesecloth and laboratory wipes are used in cleaning of radioactive wastes produced in nuclear laboratories such as the Department of Energy's Los Alamos National Laboratory (LANL). The radionuclide-contaminated cellulose wastes require suitable means of degradation and disposal. Untreated radioactive wastes with various levels of radioactive strengths are stored in large volumes over decades throughout the world necessitating disposal.Item Enzyme instigator: a portable suitcase exhibit for ninth grade biology(2009-09-19) McArthur, Brenda Harrison; Calver, Lewis E.The goal of this project was to create a portable science suitcase exhibit that ninth grade biology teachers can utilize when teaching students the complex concepts of enzymes. I created a homework handout, animation, game, hands-on models, laboratory experiments and an easy to use instruction manual. This suitcase was created to bridge the gaps between required information for standardized testing and the details presented to them in current text books. The entire suitcase was evaluated by current ninth grade biology teachers for its ease and usefulness inside their classes. The suitcase's influence on students' interest, performance and retention will be tested by DISD, STARS, and APS once the suitcase has been incorporated into the curriculum.Item Enzymes in COG2159 of the Amidohydrolase Superfamily: Structure and Mechanism of 5-Carboxyvanillate Decarboxylase (LIGW)(2015-04-06) Vladimirova, Anna VCOG2159 of the Amidohydrolase Superfamily (AHS) is composed of a wide range of enzymes, which catalyze hydration, hydrolysis, and decarboxylation reactions. 5-Carboxyvanillate decarboxylase (LigW) belongs to COG2159 and catalyzes the conversion of 5-carboxyvanillate (5-CV) to vanillate (VAN) in the pathway for the degradation of lignin. The recombinant genes from Sphingominas paucimobilis SYK-6 (LigW) and Novosphingobium aromaticivorans DSM 12444 (LigW2) were expressed in E. coli in the presence of Mn^2+ and the purified enzymes contained 1 equivalent of Mn^2+. The kinetic constants for the decarboxylation of 5-CV are as follows: kcat = 2.0 s^-1 and kcat/Km = 4.4 x 10^4 M^-1 s^-1 for the SYK-6 LigW and kcat = 27 ? 1.0 s^-1 and kcat/Km = 1.1 x 10^4 M^-1 s^-1 for the DSM 12444 LigW2. The pH-rate profiles are bell-shaped, these results are consistent with the required deprotonation of the invariant Asp-296 and protonation of His-226 for catalysis and/or substrate binding to occur. Alterations of LigW metal ligands significantly decrease the catalytic activity with kcat/Km values at least three orders of magnitude lower than that of the wild-type enzyme. Site-directed mutagenesis of substrate binding resides substantially lower or abolish the activity in LigW. The enzyme is also inhibited by the product vanillate, 3-methoxy-5-carboxybenzoate, and 4-hydroxy-3-methoxy-5-nitrobenzoate. The latter proved to be a tight binding inhibitor of LigW and LigW2 with a Ki^app = 17 nM for each of the enzymes. LigW catalyzes the exchange of the hydrogen at C-5 of VAN with deuterium over time and the product isotope effect (PIE) in a 50:50 mixture of D2O:H2O at pD 9.0 is 4.3. The crystal structures of LigW from SYK-6 and DSM 12444 with Mn^2+ in the active site were determined to a resolution of 1.8 A (PDB id: 4ICM) and 1.5 A (PDB id: 4INF). The structure of LigW was also determined complexed with the inhibitors 5-NV, MCB, and VAN. A chemical mechanism for the decarboxylation of 5-CV to VAN by LigW has been proposed. LigW requires Mn2+ for catalysis, proton transfer to C-5 is likely rate limiting for the overall reaction and precedes the decarboxylation step. The protonation of the si-face of C5 is performed from either the hydroxyl group at C4 or the carboxylate group of Asp-296. In addition, COG2159 ?-resorcylate decarboxylase (?-RSD) from Polaromonas sp. JS666 catalyzes the conversion of ?-resorcylate to resorcinol this enzyme also catalyzes the decarboxylation of 2, 4, 6- trihydroxybenzoate, 2, 3- dihydroxybenzoate, and 2, 6-dihydroxy-4-methylbenzoate.Item High throughput directed enzyme evolution using fluorescence activated cell sorting(2003-05) Olsen, Mark Jon; Iverson, Brent L.; Georgiou, GeorgeItem Immobilized metallodithiolate ligand supports for construction of bioinorganic model complexes(2009-05-15) Green, Kayla NalynnThe A-cluster active site in acetyl coA synthase exploits a Ni(CGC)2- metallopeptide as a bidentate ligand to chelate the catalytically active square-planar nickel center used to produce acetyl coA. As Nature utilizes polypeptides to isolate and stabilize the active sites, we have set out to immobilize biomimetic complexes to polyethylene-glycol (PEG) rich polystyrene polymer beads (TentaGel). The PEG rich resin-beads serve to imitate the peptidic superstructure of enzyme active sites as well as to protect the resin-bound models from O2 decomposition. As a model of the NiN2S2 ligand observed in the A-cluster of acetyl coA synthase, the CGC tripeptide was constructed on resins using Merrifield solid phase peptide synthesis and then metallated with NiII to produce bright orange beads. Derivatization with M(CO)x (M = Rh, W) provided qualitative identification of ?-Ni(CGC)M(CO)x n- via ATR-FTIR. Additionally, Neutron Activation Analysis (NAA) and UV-vis studies have determined the concentration of Ni and CGC, and qualitatively identify ?-Ni(CGC)2-. Furthermore, infrared studies and NAA experiments have been used to identify and quantify ?- Ni(CGC)Rh(CO)2 1-. The S-based reactivity of Ni(ema)2-, a good model of Ni(CGC)2-, toward oxygenation and alkylation has been pursued and compared to neutral NiN2S2 complexes. The spectroscopic, electrochemical and structural effects of these modifications will be discussed and supported using DFT computations and electrostatic potential maps of the resulting Ni(ema)*O2 2- and Ni(ema)*(CH2)3 complexes. Having firmly established the synthesis, characterization and reactivity of NiN2S2 2- systems in solution and resin-bound, CuIIN2S2 analogues were explored. The synthesis and identification of solution complexes, Cu(ema)2-, Cu(emi)2-, and Cu(CGC)2- via UV-Vis, EPR, and ?ESI-MS will be discussed in addition to their S-based reactivity with Rh(CO)2 + . Furthermore, the resin-bound Cu(CGC)2- complex has been produced and characterized by EPR and its Rh(CO)2 adduct identified by ATR-FTIR and compared to the analogous NiN2S2 2- systems. As the active site of [FeFe] Hydrogenase utilizes a unique peptide-bound propane dithiolate bridge to support the FeFe organometallic unit, [FeFe]Hydrogenase models have been covalently anchored to the resin-beads via similar carboxylic acid functionalities. The characterization (ATR-FTIR, EPR, Neutron Activation Analysis), stability and reactivity of the immobilized models complexes are discussed as well as work toward establishing the microenvironment of resin-bound complexes.Item Mechanistic studies of HPP epoxidase and DXP reductoisomerase: applications to biosynthesis and antibiotic development(2008-05) Munos, Jeffrey Wayne, 1979-; Liu, Hung-wen, 1952-The focus of this dissertation is the study of two enzymes, DXR and HppE. DXR catalyzes the first committed step in the MEP pathway, which is the pathway most eubacteria, archeabacteria, algae, and the plastids of plants use for the biosynthesis of isoprenoid. Since mammals utilize the mevalonate pathway and isoprenoids are essential for survival, all enzymes in the MEP pathway are excellent antibiotic targets. One antibiotic that has promise in the fight against malaria is the natural product fosmidomycin, whose antibiotic activity is due to its ability to bind and inhibit DXR. With a deeper understanding of DXR's catalyzed reaction, it will be possible to design a more sophisticated and potent antibiotic. To probe the mechanism of DXR, two fluorinated substrate analogues, 3F-DXP and 4F-DXP, and a fluorinated product analogue, FCH₂-MEP were designed and analyzed as possible substrates or inhibitors. To further analyze the mechanism of DXR, a 2° [²H]-KIE study was conducted using the equilibrium perturbation method. The second enzyme this dissertation examines is HppE, which catalyzes the final step in the biosynthesis of the antibiotic, fosfomycin. Fosfomycin is a clinically useful antibiotic for the treatment of limb-threatening diabetic foot infections and urinary tract infections. Chemically speaking, HppE is unique for two reasons. First, HppE's epoxidation differs from Nature's standard method of epoxide formation by alkene oxidation, where the epoxide oxygen is derived from molecular oxygen. For HppE, the epoxide is formed through the dehydrogenation of a secondary alcohol; thus the epoxide oxygen is derived from the substrate. Second, HppE is a unique member of the mononuclear non-heme iron-dependent family of enzymes. HppE differs from all other mononuclear non-heme iron-dependent enzymes by requiring NADH and an external electron mediator for turnover but not requiring [alpha]-KG, pterin, ascorbate, or an internal iron-sulfur cluster. After a study was published on the activity of zinc-reconstituted HppE from Streptomyces wedmorensis, the proposed iron and NADH dependent mechanism of HppE was reevaluated and was reconfirmed. The HppE from Pseudomonas syringae (Ps-HppE) was also purified and was characterized biochemically and spectroscopically. The results of [²H] and [¹⁸O]-KIE studies on Ps-HppE are also reported.Item Mechanistic studies of two iron-containing enzymes that catalyze unusual chemical transformations(2011-08) Chang, Wei-chen, Ph. D.; Liu, Hung-wen, 1952-; Anslyn, Eric V; Fast, Walter L; Kerwin, Sean M; Whitman, Christian PEnzymes are biological catalysts which trigger chemically inert reaction or accelerate the rates of chemical reactions, oftentimes by many orders of magnitude compared to uncatalyzed reactions. The remarkable catalytic ability afforded by enzymes derives not only from the structure and chemical properties of the enzyme active sites, but sometimes involves redox active metal ions, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species along the reaction coordiante. To enhance their catalytic ability, many enzymes have also evolved to require metal ions for activity. Metal ions adapted by enzymes often provide crucial chemical functionality and/or reactivity that are not accessible by the twenty canonical amino acids. Metal ion-containing enzymes serve to greatly broaden the diversity of chemical reactions that can be mediated by enzymes. The work described herein focuses on mechanistic studies of two enzymes that use iron to catalyze two distinctive reactions. In the first part of this work, studies will focus on the (S)-2-hydroxylpropylphosphonate epoxidase (HppE), a mono-nuclear non-heme iron containing enzyme that is an essential enzyme in fosfomycin biosynthesis, and employs an unidentified reduction system for catalysis. In biological systems, mono-nuclear non-heme iron containing enzymes mediate C-H bond activation and further diverse to various outcomes. The HppE catalyzed reaction discovered herein involves oxidative carbon-phosphorous bond migration, raising questions as chemical mechanism(s) can account for such an unusual transformation. The chemical mechanism of HppE will be interrogated with a combination of organic synthesis and biochemical techniques. Our current results suggest that the HppE may first employ a novel mode of non-heme iron containing enzymes catalysis involving hydrogen atom removal and followed by a carbocation-triggered C-P bond migration. In the second part of this dissertation, the focus is to elucidate the mechanism of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), an enzyme that plays a role in regulating the production of the isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IspH is a [4S-4Fe] enzyme that catalyzes a reductive deoxygenation reaction requiring the addition of two electrons during turnover. Although extensive efforts have been devoted to the study of this transformation, the mechanism of this intriguing reaction remains elusive. Our current data provide experimental evidence indicating the role(s) of iron-sulfur cluster and has mechanistic implication for this unusual reaction. Taken together, our studies of HppE and IspH help to illustrate the catalytic diversity of non-heme iron containing enzymes, and provide mechanistic insights into these atypical reactions.Item Model systems for the 330 nm chromophore of pyridoxal 5'- phosphate containing enzymes(Texas Tech University, 1976-08) Kang, Mohinder SinghThe absorption, fluorescence and chemical properties of the PLP site in enzymes can be simulated by model systems (9). The emphasis of this dissertation is the development of model systems for the three structures; carbinolamine, non-ionic hydrogen bonded Schiff base and substituted aldimine, proposed for the 330-335 nm chromophore of PLP containing enzymes. The PLI adducts with di- and triethylam ine were studied as models for the carbinolamine structure; the PLP adducts with t-butylarine and n-hexylamine were shown to be models for the Schiff base; and the adducts between diaminopropane and pyridine-4-aldehyde and benzaldehyde were studied as a model of the substituted aldimines structure.Chemical and physical properties of these model systems are presented and discussed in relation to PLP adducts with enzymes. Absorbance, infrared, fluorescence and NMR spectra were used to characterize the adducts. The effects of different solvents (DMSO and H„0) on PLP-amine adducts are reported and discussed.Item Novel high-throughput screening methods for the engineering of hydrolases(2011-05) Gebhard, Mark Christopher; Georgiou, George; Alper, Hal; Ellington, Andrew D.; Iverson, Brent L.; Maynard, Jennifer A.Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilitItem On the reactions of trans-3-chloroacrylic acid dehalogenase and a cis-3-chloroacrylic acid dehalogenase homologue, Cg10062 : mechanistic and evolutionary implications(2015-05) Huddleston, Jamison Parker; Whitman, Christian P.; Johnson, Kenneth A; Kerwin, Sean M; Fast, Walter L; Hoffman, David WThe tautomerase superfamily (TSF) provides an excellent model system to study enzyme specificity, catalysis, and divergent evolution. trans-3-Cholroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) are three TSF members that catalyze the final reactions in the degradation of the nematocide, 1,3-dichloropropene. All three enzymes have the TSF characteristic beta-alpha-beta fold and catalytic amino terminal proline (Pro-1). Both CaaD and cis-CaaD dehalogenate their respective isomers of 3-chloroacrylic acid yielding malonate semialdehyde. Subsequently, MSAD decarboxylates malonate semialdhyde resulting in acetaldehyde and CO2. Their catalytic and substrate specificities are exquisite considering they share three key and positionally conserved residues. As part of an effort to understand how such specificity evolved, a pre-steady-state kinetic analysis of CaaD was carried out. Alongside a similar study on cis-CaaD, a fluorescent mutant of CaaD was constructed that had minimal kinetic differences from the wild-type. The mutant was validated as an accurate fluorescent reporter of change in enzyme state that allowed for the reaction to be followed using stopped-flow methods. Stopped-flow fluorescence, rapid chemical quench data and ultraviolet spectroscopy were globally fit by computational simulation. The fit resulted in a kinetic mechanism for CaaD affording detailed information about the reaction, including measuring the rate of product release, the rate of chemistry, a previously unknown partially rate-limiting step associated with a conformational change, and the definition of binding constants for both products (MSA and Br-). In addition to the dehalogenation reaction, the reaction of the fluorescent mutant with a mechanism-based inhibitor, 3-bromopropiolate, was characterized. The values for the apparent rate of inhibition and potency were defined and estimates were determined for the values of the rate of chemistry and the release of bromide. The information gathered during these inhibition experiments was used to further refine the CaaD dehalogenation mechanism eliminating ambiguities present in the initial data set. Finally, the reactions of a cis-CaaD homologue, Cg10062 from Corynebacterium glutamicum were characterized. Cg10062 shares high sequence similarity (53%) and the same six critical active site residues as cis-CaaD, but Cg10062 has poor cis-CaaD activity. Moreover, Cg10062 dehalogenates both 3-chloroacrylic acid isomers. The reactions of Cg10062 with propiolate, 2-butynoate, and 2,3 butadienoate were investigated. Cg10062 functions as a hydratase/decarboxylase using propiolate generating malonate semialdehyde and acetaldehyde. Cg10062 catalyzes a hydration-dependent decarboxylation of propiolate as exogenously added malonate semialdehyde is not decarboxylated. With 2,3 butadienoate and 2-butynoate, Cg10062 functions as a hydratase and yields only acetoacetate. Mutations to the activating residues Glu114 and Tyr103 produced a range of results from a reduction in wild-type activity to a switch of activity. Possible intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with certain combinations of substrate and mutant enzymes. Three mechanisms are presented to explain these findings along with the strengths and weaknesses of each mechanism in terms of being able to account for experimental observations.Item Phosphorylation of hexokinase by tyrosine and serine/threonine specific kinases(Texas Tech University, 1992-12) Seale, Jeffrey WadeNot availableItem Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis(Texas Tech University, 2004-12) Sinha, ArchanaSterol methyltransferases (SMTs) are ubiquitously represented in plants and they can serve as the rate-limiting enzymes in the 24-alkyl sterol (phytosterol) pathway. Together these enzymes are capable of converting sterol acceptors with a 24(25)-double bond (cycloartenol, CA; CI-activity) or 24(28)-double bond ((24)28-methyleneIophenol, ML; -C2-activity) in the sterol side chain into more that 60 distinct phytosterols in a single plant. Recently, we discovered using the soybean SMT that depending on the nature of the substrate olefin bound to SMT, either one or two catalytic reactions can proceed, concerted or step-wise, to generate the product diversity. To investigate the proposed role of aromatic amino acids that are part of a signature motif in the active site of SMT enzymes- F82YEYGWG88, Y83L and Y83F mutants were prepared and purified to homogeneity and the steady-state kinetic parameters were determined as described in this laboratory for the wild-type soybean SMT. The Y83L mutant performed much like the wild-type enzyme in terms of substrate acceptability, product distribution and physical property, but differences were detected in Y83F mutant. "When the mutant SMT activities were compared to the native SMT activities in relation to inhibition by 25- azacycloartenol (transition state analog) or 26,27-dehydrocycloartenol (mechanism based inhibitor), both sets of enzymes were found to be inhibited with equal efficacy, suggesting that the successive C-methylation of the Ä24 bond occurs at the same active center. Based on activity assays performed over the temperature range 15 to 40''C, the activation energy (Eact in KJ/mol) estimated from the Arrhenius plots were found to be: (i) wild-type, CA = 49, ML = 71; (ii) Y83F, CA = 65, ML = 52 and (iii) Y83L, CA = 98, ML =185. Analysis of the pH dependence of log kcat/Km for the wild-type and two mutants showed different profiles for Ä 24(25) and Ä24(28) -substrates. The results of the mutational and kinetic analyses are interpreted to imply that product diversity catalyzed by the soybean SMT is made possible by the relaxed control over substrate and intermediate conformations resulting from altered cation-ð interactions in the active sites of the mutant enzyme and relates to the different mechanisms catalyzed using different olefin substrates.