Browsing by Subject "Biosynthesis"
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Item Alkaloid biosynthesis in Claviceps purpurea PRL 1980 protoplasts(Texas Tech University, 1979-12) Lu, Jean-PhonNot availableItem The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme(2008-05) Szu, Ping-Hui, 1978-; Liu, Hung-wen, 1952-D-Desosamine, a 3-(dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae, plays an essential role in conferring biological activities to its parent aglycones. The proteins encoded by the desI and desII genes in the methymycin/pikromycin biosynthetic gene cluster have been proposed to catalyze C-4 deoxygenation in D-desosamine biosynthesis. DesI is a pyridoxal 5'-phosphate-dependent C4-aminotransferase and catalyzes a transamination reaction converting thymidine diphosphate (TDP)-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-glucose. DesII, which contains a [4Fe-4S] cluster binding motif, CXXXCXXC, has been identified as a member of the radical S-adenosylmethionine (SAM) enzyme superfamily by sequence analysis. To study the catalytic function of DesII, the desII gene was heterologously overexpressed in Escherichia coli and the DesII protein was purified to near homogeneity. Biochemical studies clearly established that the substrate for DesII is TDP-4-amino-4,6-dideoxy-D-glucose, and DesI and DesII function independently to carry out C-4 deoxygenation. DesII requires a [4Fe-4S]¹⁺ center and Sadenosylmethionine for activity. Accordingly, the originally proposed mechanism for C-4 deoxygenation in which DesI and DesII function together was revised. Two possible mechanisms have subsequently been proposed for the DesII reaction. The DesII catalysis is likely initiated by the formation of a 5'-deoxyadenosyl radical followed by the C-3 hydrogen atom abstraction. In the first possible route, the key step is a radical-induced deamination followed by the readdition of ammonia to the resulting cation radical intermediate, which is effectively a 1,2-amino shift, to form an aminol radical. Alternatively, the reaction may involve deprotonation of the 3-hydroxyl group to yield a ketyl radical anion to facilitate the [beta]-elimination of the ammonia group. Interestingly, DesII is flexible towards TDP-D-quinovose and TDP-3-amino-3,6-dideoxy-D-glucose. A possible biological reducing system, flavodoxin, flavodoxin reductase, and NADPH, for the reduction of the [4Fe-4S]²⁺ cluster, was also identified. Deuterium incorporation into SAM using C-3 deuterium-labeled substrate provides solid evidence for C-3 hydrogen atom abstraction by the 5'-deoxyadenosyl radical in the proposed mechanism. TDP-3-fluoro-3,6-dideoxy-D-glucose serves as a competitive inhibitor for DesII, which is in favor of deprotonation of the C-3 hydroxyl group being involved in DesII catalysis.Item Biosynthetic studies of thiosugar-containing natural products, BE-7585A and Lincomycin A(2011-05) Sasaki, Eita; Liu, Hung-wen, 1952-; Anslyn, Eric V; Bielawski, Christopher; Fast, Walter L; Whitman, Christian PSulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been well understood. On the other hand, a wide variety of sugar derivatives commonly found in natural products are often vital components for the efficacy and specificity of their parent molecules. Elucidation of such unusual sugar biosyntheses is important both for understanding their intriguing chemical mechanisms and creating unnatural compounds by altering their biosynthetic machineries, which could potentially exhibit enhanced or novel biological activities. This dissertation describes biosynthetic studies of two thiosugar-containing natural products, BE-7585A and lincomycin A, produced by Amycolatopsis orientalis and Streptomyces lincolnensis, respectively. While the former possess a C-2-thiosugar-containing disaccharide moiety, the latter contains a C-1-thio substituent on a characteristic eight-carbon backbone sugar. The focus of this research is to characterize the biological pathways and mechanisms responsible for the sulfur incorporation and the unique sugar scaffolds. BE-7585A, an angucycline-type natural product, contains the rare C-2-thiosugar moiety. PCR-based screening of a cosmid library constructed from the genomic DNA of A. orientalis led to the identification of the BE-7585A biosynthetic gene cluster. A gene, bexX, was found to be a candidate for a thiosugar synthase with moderate sequence similarity to a thiazole synthase. The gene, bexX, and a glycosyltransferase homologue, bexG2, were heterologously expressed in Escherichia coli. A variety of biochemical experiments provided a wealth of evidence supporting the proposed biosynthetic pathway for the C-2-thiodisaccharide moiety. Finally, whole genome sequencing and a genome mining approach led to the identification of a sulfur carrier protein to accomplish the in vitro enzymatic synthesis of the C-2-thiosugar for the first time. Lincomycin A is a lincosamide antimicrobial natural product with a C-1 methylthio substituent. Although the lincomycin A biosynthetic gene cluster has been reported, biochemical verification of the biosynthetic pathway has remained elusive. In this dissertation, the complete methlthiolincosamide biosynthetic pathway including the potential C-1 sulfur incorporation mechanism was proposed. Furthermore, two early intermediates of the pathway were characterized for the first time by demonstrating the LmbR (transaldolase) and LmbN (isomerase) reactions in vitro.Item Design and synthesis of benzo-18-crown-6 carboxylic acids and diazadibenzo crown ethers and lariat ethers with neutral and proton-ionizable side arms(Texas Tech University, 2001-12) Kim, MyeongseobA series of proton-ionizable benzo-18-crown-6 lariat ethers has been prepared to probe the optimal host structure for a ferrioxamine B guest. Host-guest supramolecular assembly formation constants involving second-sphere complexation of the siderophore ferrioxamine B by a lariat ether carboxylic acid host were obtained from liquid-liquid extractions at high and low pH. The formation constant, Ka, determined at pH = 3.2 is compared to those from other lariat ether carboxylic acids of different chain lengths. These constants are similar to the parent crown ether. At pH = 9.3, the lariat ethers are ionized and this results in a more stable complexes. According to the comparison of formation constants at pH =9.3, an optimal host structure for the complexation of ferrioxamine B guest is determined. To study the complexation behavior of nitrogen containing macrocyclic ligands toward transition metal cations, series of diazadibenzocrown ethers and diazadibenzo lariat ethers with neutral side arms and proton-ionizable side arms were synthesized. Solvent extraction and membrane transport studies were performed with 1,13- diazadibenzo-15-crown-5, 1,13-diazadibenzo-18-crovra-6, 1,13-diazadi(?er^butylbenzo)- 18-crown-6 and l,13-diazadibenzo-18-crown-6 diester and showed that 1,13-diazadi (?ert-butylbenzo)-18-crown-6 and a l,13-diazadibenzo-18-crown-6 diester selectively forms complexes with Pb^2+ in the presence of Cd^2+ and Zn^2+.Item Developing a diagnostic tool for acyl carrier proteins through trypsinolysis, reverse-phase chromatography and native chemical ligation(2010-08) Reyes, Graciela, 1957-; Willets, Katherine A.; Keatinge-Clay, Adrian TristanPolyketide biosynthesis is a field that has had tremendous advances in the past 50 years. The understanding of the mechanisms is updated as investigations delve into domain interactions of these microbial natural products. Although numerous polyketides are known, similarities in the sequence of product generation can be used as templates for further exploration of enzymatic activity. The focus of studies recently has been towards developing protocols to manipulate the natural products resulting in medicinally important manufactured products. This investigation examined the mechanism of the acyl carrier protein (ACP) module involved in biosynthesis.Item Enzymatic features and biocatalytic applications of modular polyketide synthase domains(2015-12) Bailey, Constance Beryl; Keatinge-Clay, Adrian Tristan; Anslyn, Eric V; Liu, Hung-Wen; Hoffman, David; Garneau-Tsodikova, SylviePolyketides are a class of secondary metabolites that are notable for their chemical diversity and therapeutic relevance. They are biosynthesized by polyketide synthases (PKSs) megasynthase enzymes in an assembly-line fashion. Though the molecular architectures of polyketides are complex, their biological precursors are chemically simple. Thus, understanding this powerful biosynthetic machinery is of interest for synthetic biology and biocatalytic applications. This dissertation presents three projects that decipher underlying mechanistic features and explore biocatalytic applications of PKSs. In modular PKSs, one module corresponds to one round of keto-elongation followed by modification through the action of β-carbon processing domains. The first project employs a system wherein a single module is used in vitro to generate small, chiral PKS products (triketide lactones). Although triketide lactones are a common output for PKS enzymology assays, usually they are only observed in trace quantities. In this study, we performed a number of strategies to scale up the production of triketide lactones to facilitate their use as chiral building blocks for chemical synthesis. In this process, we also gained new insights regarding the interacting kinetics and selectivities of the domains in an in vitro environment. ix The second project focused on the ketoreductase (KR) domain, which sets the majority of the stereogenic centers within a polyketide, and thus has obvious potential for biocatalytic applications. This project employs a structure-activity relationship (SAR)- type approach to dissecting stereocontrol. The SAR results, in concert with crystallographic data inspired two rational mutations that were sufficient to reverse the stereoselectivity of a representative KR. Thus, we were able to employ a rational approach to engineering stereocontrol. The final project also focuses on the KR domain, however from a subclass of PKSs termed trans-acyltranferase (AT) PKSs. In contrast to the canonical cis-AT PKSs, the trans-AT PKSs have more varied modular organizations and architectures. One of these peculiar organizations one termed a “split” bimodule, wherein domains within a module are present on different polypeptides. Structural characterization of a KR from a split bimodule revealed features that may correspond to interpeptide interactions that afford communication between the two polypeptides of the split bimodule. Additionally, bioinformatic analysis of KRs from split bimodules reveals a number of diagnostic sequence motifs.Item Factors affecting poly-b-hydroxybutyrate synthesis and crystal formation in Bacillus thuringiensis(Texas Tech University, 1984-08) Hickerson, Steven LynnNot availableItem Functional characteristics of genes involved in brassinosteroid signaling in cotton(Texas Tech University, 2003-12) Sun, YanCotton fibers are highly elongated single celled trichomes that grow from the seed integument. Elongation of fiber cells begins almost after ovule fertilization and continues for approximately 20 days. In this study, I have shown that BR signaling is necessary for cotton fiber initiation and elongation, using in vitro cultured cotton ovule with Brassinosteroids (BR) and brassinazole (Brz, BR biosynthesis inhibitor). BRs are polyhydroxylated sterol derivatives of plant origin that are required for normal plant development. Several Arabidopsis genes that encode critical components of this pathway have been identified through genetic screening. BRM encodes a membrane-bound leucine-rich receptor-like kinase that apparently acts as the BR receptor. BIN2, which acts downstream of BRM in this pathway, encodes a GSK3/SHAGGY-like kinase that down-regulates BR signaling. To understand the role of cotton orthologous genes in fiber development, cotton ESTs similar to the Arabidopsis BRM and BIN2 genes were identified. These ESTs were used to clone the corresponding full-length GhBRM and GhBIN2 cDNAs. The GhBRM was cloned from a cotton cDNA library and then amplified from cotton genomic DNA. This 3561 bp gene contains no introns and encodes a protein with 1187 amino acids. Database analyses shows that the GhBRM protein has all the distinct domains characteristic of BRM. Four GhBIN2 cDNAs were also cloned. They all include a coding sequence of 1146 base pairs in length and encode derived proteins of 381 amino acids. Sequence comparison with mammalian GSK3p and Drosophila GSK3/SHAGGY-like kinase showed that GhBIN2 proteins share many conservative regions with these two GSK3/SHAGGY-like kinases. Analysis of the expression patterns of the GhBRM and GhBIN2s genes using quantitative real-time PCR showed that they are expressed throughout cotton plants, including leaves, buds, hypocotyls, roots, sepals, ovules, bolls, and fibers. To identify the functions of these genes, gene constructs that express GhBRM and GhBIN2 under control of a CaMV 35S promoter were developed. Expression of the GhBRM transgene in the dwarf bri1-5 mutant Arabidopsis plants restored them to normal height. Expression analysis showed that the heights of the transgenic plants were significantly correlated with the GhBRH expression level (r = 0.97). These results strongly suggest that the GhBRH gene encodes a functional BR receptor protein. Conversely, expression of the GhBIN2 transgene in wildtype Arabidopsis plants resulted in severe stunting similar to strong BR deficient or insensitive mutants. Expression analysis showed that the heights of the transgenic plants were inversely correlated with these GhBlN2 expression levels (the average correlation level r= -0.90). These results indicate that the GhBlN2 genes function as negative regulators of BR signal transduction pathway. These results confirm that the GhBRM and GhBIN2 cDNAs encode proteins that are capable of functioning in the BR signaling pathway. BR signal transduction pathway could provide the basis for genetic modification of fiber development.Item Genetic and biochemical studies of the biosynthesis and attachment of D-desosamine, the deoxy sugar component of macrolide antibiotics produced by Streptomyces venezuelae(2004) Borisova, Svetlana Alekseyevna, 1976-; Liu, Hung-wen, 1952-Macrolide antibiotics are clinically important drugs widely used to treat the infections caused by gram-positive bacteria. They consist of a macrolactone aglycone unit and one or more deoxy sugar component(s). The presence of an at least one deoxy sugar moiety in the structure of macrolides is essential for their antimicrobial activity. Modifications of the deoxy sugar substituent hold promise as a valuable approach towards generating new macrolide antibiotics with improved biological properties. The development of new antimicrobial drugs is essential to combat the growing problem of the pathogen resistance to the existing antibiotics. This thesis describes a part of our ongoing effort to investigate the mechanistic details of the biosynthetic pathway to D-desosamine, an amino deoxy sugar component of macrolide antibiotics methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae. D- Desosamine also exists in many other clinically important macrolides, e.g. erythromycin, clarithromycin, and oleandomycin. In particular, the gene knockout technique was used to explore the functions of the desI, desII, and desVIII genes of the D-desosamine biosynthetic gene cluster. The analysis of the macrolides produced by the resulting S. venezuelae mutants has led to the revision of the originally proposed pathway to desosamine. A new mechanism for the C-4 deoxygenation step was also envisioned. Four new macrolides were isolated in these studies, which established the relaxed substrate specificity of the glycosyl- transferase DesVII involved in the coupling of TDP-sugar derivatives to the aglycone in the pathway. To further explore the potential of DesVII to couple various sugar and aglycone substrates, desVII was expressed in E. coli and the recombinant DesVII protein was used to study the in vitro glycosyltransferase activity. Our results demonstrate that DesVII requires an additional protein component, DesVIII, to perform the catalysis, and the activity is optimal at pH 9. These conditions, unusual for the known glycosyl transfer reactions, may prove to be the general requirements for other macrolide glycosyltransferases. The preliminary study of the substrate specificity of the DesVII/DesVIII catalytic pair was conducted in vitro and showed the potential for their application to glycosylation in combinatorial biosynthesis.Item Hydroxylation of intermediates in clavine alkaloid biosynthesis.(Texas Tech University, 1974-08) Bajwa, Raghbir SinghNot availableItem In vitro polyketide biocatalysis : triketide building-blocks and enzymology(2013-05) Harper, Andrew David; Keatinge-Clay, Adrian TristanPolyketide products are useful compounds to research and industry but can be difficult to access due to their richness in stereogenic centers. Type I polyketide synthases offer unique engineering opportunities to access natural stereocontrol and resultant complex compounds. The development of a controlled in vitro platform based around type I polyketide synthases is described. It has been used to produce a small library of polyketide fragments on an unprecedented and synthetically-relevant scale and explore polyketide synthase enzymology.Item Inducible plant responses triggered by phytochemical and bacterial elicitors(Texas Tech University, 2004-05) Farag, Mohamed Ali AliPlants release elevated levels of volatile organic compounds (VOCs) in response to insect or mechanical damage with some volatile components serving as chemical signals for attracting or repelling other organisms. Plants exposed to biogenic VOCs present in the environment can also modulate plant growth and development, although the nature of VOC-triggered plant responses has yet to be characterized for most plant species. The overall goal of this research has been to examine how certain environmental VOCs can trigger primary and secondary responses in plants. Specifically we examined (1) metabolic changes triggered by Ce alcohols and aldehydes ubiquitously released from damaged plants focusing on the model systems tomato and maize, as well as (2) C4 alcohols emitted from plant growth promoting rhizobacteria in the classic model plant Arabidopsis. Metabolic changes were monitored at genomic levels by reverse transcriptase-PCR (RT-PCR), slot blot analysis and GUS fusion assays. Changes in metabolic levels were monitored by GC, HPLC and Western analyses. The Ce-volatile (£)-2-hexenal triggered the release of local and systemic mono-and sesquiterpenes in tomato which was shown to be mediated through the jasmonic acid (JA) signaling pathway. Release of VOCs triggered with Ce-volatile treatment did not affect the accumulation of proteinase inhibitor enzymes (PI) or stored phytochemicals. In maize, Ce-com volafile (Z)-3-hexenoI resulted in an increase in transcript level for a series of defense genes including pal (phenyl alanine ammonium lyase), lox (lipoxygenase), igl (indole-3-glycerol phosphate lyase) and mpi (maize-proteinase inhibitor). The induction of transcripts were compared with metabolites generated from the respective pathways. Structure activity relationships has established that a series of Cft- alcohols were more active than tested aldehydes in triggering VOC emissions in both model systems. Biochemical and plant assays showed that the bacterial volatile elicitor 2,3- butanediol triggered growth promotion and induced disease resistance in Arabidopsis. Using transgenic and mutant lines of Arabidopsis, the signal pathway activated by bacterial volatiles was found to be dependent on cytokinin activation for growth promotion and dependent on ethylene signaling for induced pathogen resistance. These data provide new insight into the role of short chain alcohols and aldehydes as signaling molecules mediating plant-plant or plant-microbial interactions.Item Inside the microbial weapons factory: structural studies of polyketide biosynthetic machinery(2015-08) Gay, Darren Christian; Keatinge-Clay, Adrian Tristan; Robertus, Jon; Hoffman, Dave; Barrick, Jeff; Ekerdt, JohnPolyketides are a class of small molecules synthesized by a broad spectrum of bacteria, plants, and fungi, and many exhibit powerful bioactive properties. The number of clinically-relevant compounds adapted from polyketide scaffolds is growing, eliciting attempts from synthetic organic chemists to construct polyketide-related compounds in the laboratory from simple chemical building blocks. Unfortunately, the current efficiency by which a skilled artisan can synthesize even small quantities of a polyketide is severely limited by the functional and stereochemical complexity of these compounds. Conceptually, it would be much simpler to genetically reprogram the enzymes responsible for polyketide biosynthesis to produce designer molecules; however, the massive size of polyketide synthase enzymes has hindered efforts towards understanding critical features of their structures and mechanisms. Only very recently has structural information become available for enzymes involved in polyketide biosynthesis, providing an initial glimpse into the inner workings of these subcellular pharmaceutical factories. It will not be possible for mankind to fully realize the potential of engineered polyketide synthases without understanding how their architectures govern the molecules they have evolved to produce. In this work, the structure and mechanism of several enzymes involved in polyketide biosynthesis is investigated. An unprecedented architecture for the ketoreductase-enoylreductase didomain from the second module of the spinosyn polyketide synthase reveals structural divergence from the related mammalian fatty acid synthase, and reconstituted in vitro activity of the enoylreductase domain indicates the isolated enzyme retains activity apart from its parent polyketide synthase module. The dehydratase domain isolated from the tenth module of the rifamycin polyketide synthase, previously hypothesized to only form double bonds with (Z) geometry, was found to have altered stereoselectivity dependent on the carrier handle bound to the substrate. The enoyl-isomerase domain, isolated from the fourteenth module of the bacillaene polyketide synthase, utilizes a catalytic mechanism that relies only on a single active site histidine. A series of ketosynthase domains from trans-acyltransferase polyketide synthases reveal how polyketides bind covalently to the active site of the ketosynthase, and how the flanking subdomain of the ketosynthase is used as an anchor point for the assembly of the polyketide synthase megacomplex.Item Investigation and engineering of macrolide antibiotic sugar biosynthesis and glycosylation pathways of actinomycetes(2006) Melançon, Charles Evans, 1975-; Liu, Hung-wen, 1952-Item Investigation of the post-polyketide synthase (PKS) modifications during spinosyn A biosynthesis in Saccharopolyspora spinosa(2010-08) Kim, Hak Joong; Liu, Hung-wen, 1952-Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis.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 Metabolism of 4-Dimethylallyltryptophan in Claviceps purpurea PRL 1980(Texas Tech University, 1975-05) Saini, Mohan SinghNot availableItem Probing the Biosynthesis and Mode of Action of Azinomycin B(2010-10-12) Kelly, Gilbert ThomsonSince the isolation of azinomycins A and B in 1954 from the soil bacterium, Streptomyces sahachiroi, these natural products have been synthetic targets. Both compounds exhibit in vitro cytotoxic activity at submicromolar levels and demonstrate anti-tumor activities comparable to that of mitomycin C in vivo. Unique to this class of natural products is the presence of an aziridine [1,2-a] pyrrolidine ring system. Coupled with an epoxide moiety, these structural functionalities impart the ability to form interstrand cross-links with DNA via the electrophilic C10 and C21 carbons of azinomycin and the N7 positions of suitably disposed purine bases. This dissertation investigates the global impact of azinomycin B treatment in a yeast model with special emphasis on DNA damage response, the resulting cell cycle effects, and cellular localization of the compound. The results provide the first demonstration of the in vivo actions of azinomycin B and are consistent with the proposed role of the drug as a DNA crosslinking agent. Biosynthesis of azinomycin B was investigated and appears to have polyketide, non-ribosomal peptide synthetase and alkaloid origins. In pursuit of elucidating the biosynthetic origin we developed both a cell culturing system and a cell-free extract procedure capable of supporting azinomycin synthesis; we used these. These were employed with labeled metabolites to probe the biosynthetic origins of the molecule. Investigations with this enzyme preparation imparted important information regarding the substrate and cofactor requirements of the pathway. These results supported the premise of a mixed origin for the biosynthesis of the molecule and paved the way for expansive stable isotope labeling studies, which largely revealed the biosynthetic precursors and probable construction of the azinomycins. Some of these studies corroborate while other results conflict with initial proposed biosynthetic routes based upon the azinomycin biosynthetic gene cluster sequence. Future azinomycin biosynthetic gene cluster enzyme characterization, mechanistic investigations, and genetic modifications will ultimately provide definitive proof for the intermediacy of proposed biosynthetic precursors and the involvement of specific cofactors. Better understanding of how nature constructs unique molecule may provide insight into eventual chemoenzymatic/gene thearapy based approaches toward cancer therapy.Item Profiling the natural products from the cancer-plant Sutherlandia frutescens reveal disrupted sterol homeostasis(2013-08) Thomas, Crista D.; Nes, William David; Shaw, Robert W.From the non-saponifiable fraction of the “cancer bush”- Sutherlandia frutescens was isolated 12 sterols and a pentacyclice triterpenoid mixture of a- and b-amyrin. These natural products derived from the isoprenoid biosynthesis pathway were characterized by a combination of chromatographic and spectral methods. The major sterols of physiological significance to accumulate in stems and leaves at 1% dry weight of original sample were cycloartenol and sitosterol in a ratio of approximately 1 to 1. The notably high accumulation of cycloartenol in this plant is very uncommon but nonetheless is at a level which coincides with the high level of the cancer preventing cycloartane glycosides known as sutherlandiosides. We surmise that developmental of regulation of phytosterol biosynthesis has occurred to block cycloartenol conversion to sitosterol affording redirection of cycloartenol into the glycoside derivatives.Item Regulation of arabidopsis trichome patterning and anthocyanin biosynthesis by the TTG1-bHLH-MYB complex(2007) Zhao, Mingzhe, 1973-; Lloyd, Alan M.A network of three classes of proteins consisting of bHLH and MYB transcription factors and a WD40 repeat protein - TRANSPARENT TESTA GLABRA1 (TTG1) act in concert to activate trichome initiation and patterning in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while CPC does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.