Fusarium mycotoxin-contaminated wheat containing deoxynivalenol alters the gene expression in the liver and the jejunum of broilers

The effects of mycotoxins in the production of animal feed were investigated using broiler chickens. For the feeding trial, naturally Fusarium mycotoxin-contaminated wheat was used, which mainly contained deoxynivalenol (DON). The main effects of DON are reduction of the feed intake and reduced weight gain of broilers. At the molecular level, DON binds to the 60 S ribosomal subunit and subsequently inhibits protein synthesis at the translational level. However, little is known about other effects of DON, for example, at the transcriptional level. Therefore, a microarray analysis was performed, which allows the investigation of thousands of transcripts in one experiment. In the experiment, 20 broilers were separated into four groups of five broilers each at day 1 after hatching. The diets consisted of a control diet and three diets with calculated, moderate concentrations of 1.0, 2.5 and 5.0 mg DON/kg feed, which was attained by exchanging uncontaminated wheat with naturally mycotoxin-contaminated wheat up to the intended DON concentration. The broilers were held at standard conditions for 23 days. Three microarrays were used per group to determine the significant alterations of the gene expression in the liver (P < 0.05), and qPCR was performed on the liver and the jejunum to verify the results. No significant difference in BW, feed intake or feed conversion rate was observed. The nutrient uptake into the hepatic and jejunal cells seemed to be influenced by genes: SLC2A5 (fc: −1.54, DON2.5), which facilitates glucose and fructose transport and SLC7A10 (fc: +1.49, DON5), a transporter of d-serine and other neutral amino acids. In the jejunum, the palmitate transport might be altered by SLC27A4 (fc: −1.87, DON5) and monocarboxylates uptake by SLC16A1 (fc: −1.47, DON5). The alterations of the SLC gene expression may explain the reduced weight gain of broilers chronically exposed to DON-contaminated wheat. The decreased expressions of EIF2AK3 (fc: −1.29, DON2.5/5) and DNAJC3 (fc: −1.44, DON2.5) seem to be related to the translation inhibition. The binding of DON to the 60 S ribosomal subunit and the subsequent translation inhibition might be counterbalanced by the downregulation of EIF2AK3 and DNAJC3. The genes PARP1, MPG, EME1, XPAC, RIF1 and CHAF1B are mainly related to single-strand DNA modifications and showed an increased expression in the group with 5 mg DON/kg feed. The results indicate that significantly altered gene expression was already occurring at 2.5 mg DON/kg fee

Elucidation of the structure and mechanism of the MtrCDE multi drug transporter in Neisseria gonorrhoeae

The Mtr (Multiple transferable resistance) transport system in Neisseria gonorrhoeae was found to confer the resistance of gonococci to penicillin and structural diverse hydrophobic agents (HAs), such as drugs, dyes, detergents and host-derived compounds (fatty acids and bile salts), as well as some cationic antimicrobial peptides. The mtr operon encodes an energy-dependant efflux pump system and this MtrC-MtrD-MtrE system is negatively regulated by the transcriptional repressor MtrR. The first part of this thesis presents insights into a structural study of the two membrane components, the inner membrane protein MtrD and the outer membrane protein MtrE. MtrD and MtrE have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Unfortunately, due to the difficulty of membrane protein crystallography, the best resolution of the MtrE crystals was only 8A and of the MtrD crystals was 20Å, so the structures of both MtrE and MtrD haven't been solved yet. However, this preliminary X-ray analysis leads to the possibility of solving these structures in the near future. In the mean time, using pull-down assay, growth curve analyses and ITC( Isothermal Titration Calorimetry), we established that the antiporter MtrD, the adaptor MtrC and the outer membrane protein MtrE form a contiguous complex. Assembly of the pump is constitutive, even in absence of substrate. Without the assistance of MtrC, MtrD was unable to export its substrate, Nafcillin, and the efficiency of this assembly could be enhanced by the presence of the outer membrane protein MtrE. MtrD could interact with MtrE independently in pull-down assays, showing they can form a complex even in the absence of MtrC. This behaviour is consistent with the increased hypersensitivity to Vancomycin of the recombinant strain expressing MtrE-MtrD, which indicated that MtrE could be opened by MtrD in the presence of the substrate. However, the interaction between them was not detected by ITC, suggesting this interaction is weak or energetically unfavourable. The a-helical hairpin domain of MtrC was also over expressed and purified to test for its interaction with MtrE or MlrD. Fascinatingly, the cross-linked hairpin formed a hexamer and AFM (Atomic Force Microscopy) studies revealed it arranged into a cylindrical structure. In addition, ITC studies revealed that the hairpin domain could bind to both MtrE and MtrD, suggesting that MtrC might form a channel, one end of which interacts with MtrD and the other with MtrE. Growth curves also showed that the periplasmic hairpin domain could enhance the transport activity of MtrCDE, but couldn't activate the transport of MlrD. indicating it probably works by stabilizeing the open form of MtrE. The expression of mtrCDE is believed to be under the control of a TetR-type transcriptional regulator repressor, mtrR. MtrR foims a dimer but with presence of dsDNA, it forms a tetramer. Fragments of the antimicrobial polypeptide LL-37 were synthesized and tested by titrating to MtrR by ITC. The C-tenninal of LL-37(29-37) is the part that binds to MtrR and N-terminal (I-II) is not. Interestingly, interaction of MtrR with Penicillin G demonstrated for first time that MtrR might work as a ß-lactamase; with its enzyme activity reduced after mutanting His l05 to Tyr, which was reported to be found naturally in penicillin sensitive N, gonorrhoeae strains

Lysosomaler Transport kationischer Aminosäuren durch Mitglieder der SLC7-Familie

Lysosomaler Transport kationischer Aminosäuren (KAS) stellt einen Rettungsweg in der Cystinose-Therapie dar. Ein solches Transportsystem wurde in humanen Hautfibroblasten beschrieben und mit System c benannt. Des Weiteren stellt lysosomales Arginin eine Substratquelle für die endotheliale NO-Synthase (eNOS) dar. Das von der eNOS gebildete NO ist ein wichtiges vasoprotektiv wirkendes Signalmolekül. Ziel war es daher, herauszufinden, ob Mitglieder der SLC7-Unterfamilie hCAT möglicherweise System c repräsentieren.rnIn dieser Arbeit konnte ich die lysosomale Lokalisation verschiedener endogener, sowie als EGFP-Fusionsproteine überexprimierter CAT-Isoformen nachweisen. Mittels Fluoreszenz-mikroskopie wurde festgestellt, dass die in U373MG-Zellen überexprimierten Fusionsproteine hCAT-1.EGFP sowie SLC7A14.EGFP mit dem lysosomalen Fluoreszenz-Farbstoff LysoTracker co-lokalisieren. Eine Lokalisation in Mitochondrien oder dem endoplasmatischem Retikulum konnte mit entsprechenden Fluoreszenz-Farbstoffen ausgeschlossen werden. Zusätzlich reicherten sich die überexprimierten Proteine hCAT-1.EGFP, hCAT-2B.EGFP und SLC7A14.EGFP in der lysosomalen Fraktion C aus U373MG-Zellen zusammen mit den lysosomalen Markern LAMP-1 und Cathepsin D an. Gleiches galt für den endogenen hCAT-1 in der lysosomalen Fraktion C aus EA.hy926- und U373MG-Zellen sowie für den SLC7A14 in den humanen Hautfibroblasten FCys5. Mit dem im Rahmen dieser Arbeit generierte Antikörper gegen natives SLC7A14 konnte erstmals die endogene Expression und Lokalisation von SLC7A14 in verschiedenen Zelltypen analysiert werden.rnObwohl eine Herunterregulation des hCAT-1 in EA.hy926-Endothelzellen nicht zu einer Reduktion der Versorgung der eNOS mit lysosomalem Arginin führte, ist eine Funktion von hCAT-1 im Lysosom wahrscheinlich. Sowohl die [3H]Arginin- als auch die [3H]Lysin-Aufnahme der Fraktion C aus U373MG-hCAT-1.EGFP war signifikant höher als in die Fraktion C aus EGFP-Kontrollzellen. Dies konnte ebenfalls für den hCAT-2B.EGFP gezeigt werden. Zusätzlich zeigten lysosomale Proben aus U373MG-hCAT-2B.EGFP-Zellen in der SSM-basierten Elektrophysiologie eine elektrogene Transportaktivität für Arginin. Das Protein SLC7A14.EGFP zeigte in keiner der beiden durchgeführten Transportstudien eine Aktivität. Dies war unerwartet, da die aus der Diplomarbeit stammende und im Rahmen dieser Dissertation erweiterte Charakterisierung der hCAT-2/A14_BK-Chimäre, die die „funktionelle Domäne“ des SLC7A14 im Rückgrat des hCAT-2 trug, zuvor den Verdacht erhärtet hatte, dass SLC7A14 ein lysosomal lokalisierter Transporter für KAS sein könnte. Diese Studien zeigten allerding erstmals, dass die „funktionelle Domäne“ der hCATs die pH-Abhängigkeit vermittelt und eine Rolle in der Substraterkennung spielt.rnZukünftig soll weiter versucht werden auch endogen eine Transportaktivität der hCATs für KAS im Lysosom nachzuweisen und das Substrat für das intrazellulär lokalisierte Waisen-Protein SLC7A14 zu finden. Eine mögliche Rolle könnte SLC7A14 als Transporter für Neurotransmitter spielen, da eine sehr prominente Expression im ZNS festgestellt wurde.rnLysosomal transport of cationic amino acids (CAS) constitutes a salvage pathway in the therapy of cystinosis. Such a transport system has been described in human skin fibroblasts and named system c. Furthermore lysosomal arginine plays a role in substrate supply of the endothelial NO synthase (eNOS). NO generated by cytosolic eNOS is an important vasoprotective signaling molecule. Objective of this work was to find out, if members of the SLC7 subfamily of human cationic amino acid transporters (hCATs) might represent system c.rnIn this study I could demonstrate the lysosomal localization of several endogenous and as EGFP fusion proteins overexpressed hCAT isoforms. Fluorescence microscopy revealed, that the fusion proteins hCAT-1.EGFP and SLC7A14.EGFP co-localized with the lysosomal fluorescent dye LysoTracker when overexpressed in U373MG cells. Localization in mitochondria or the endoplasmic reticulum could be excluded using appropriate fluorescent dyes. In addition the overexpressed proteins hCAT-1.EGFP, hCAT-2B.EGFP und SLC7A14.EGFP enriched in the lysosomal fraction C derived from U373MG cells, together with the lysosomal markers LAMP-1 und Cathepsin D an. The same was true for endogenous hCAT-1 in the lysosomal fraction C derived from EA.hy926 and native U373MG cells and for SLC7A14 in human skin fibroblasts. The antibody against native SLC7A14, which has been generated during this work, has enabled us to study endogenous expression and localization of SLC7A14 in several cell types for the first time.rnAlthough down regulation of hCAT-1 in EA.hy926 endothelial cells did not lead to a reduction of the lysosomal arginine supply of eNOS, a function of hCAT-1 in the lysosome is most likely. Uptake of [3H]arginine as well as [3H]lysine of fraction C derived from U373MG-hCAT-1.EGFP was significantly higher than of fraction C derived from EGFP control cells. Similar results were obtained for hCAT-2B.EGFP. Additionally, lysosomal membranes derived from U373MG-hCAT-2B.EGFP cells exhibited electrogenic transport activity for arginine using SSM-based electrophysiology. SLC7A14.EGFP did not show any transport activity in both experimental setups. This was unexpected, because the chimeric protein hCAT-2/A14_BK, which has been created during my diploma thesis and its characterization has been broadened during my dissertation did transport arginine with similar but not identical properties as hCAT-2A. The chimera carries the “functional domain” of SLC7A14 in the backbone of hCAT-2 and thus substantiated the suspicion that SLC7A14 is a lysosomal localized CAS transporter. However, these studies revealed, that the „functional domain“ of hCATs mediates pH dependence, additionally to substrate affinity and trans-stimulation properties and is involved in substrate recognition.rnProspective studies should now focus on the detection of endogenous transport activity of hCATs in the lysosome and on the identification of the substrate for SLC7A14. A role of SLC7A14 as transporter for neurotransmitter is possible, because a prominent mRNA expression of this SLC7 member has been detected in the central nervous system.rn159 S

The role of airway mucus in pulmonary toxicology.

Airway mucus is a complex airway secretion whose primary function as part of the mucociliary transport mechanism is to to serve as renewable and transportable barrier against inhaled particulates and toxic agents. The rheologic properties necessary for this function are imparted by glycoproteins, or mucins. Some respiratory disease states, e.g., asthma, cystic fibrosis, and bronchitis, are characterized by quantitative and qualitative changes in mucus biosynthesis that contribute to pulmonary pathology. Similar alterations in various aspects of mucin biochemistry and biophysics, leading to mucus hypersecretion and altered mucus rheology, result from inhalation of certain air pollutants, such as ozone, sulfur dioxide, nitrogen dioxide, and cigarette smoke. The consequences of these pollutant-induced alterations in mucus biology are discussed in the context of pulmonary pathophysiology and toxicology

Expressão genética de Gase, GSase e GDH em Misgurnus anguillicaudatus

Mestrado em Biologia MarinhaMuitos peixes conseguem sobreviver a elevados níveis de amónia ambiental; entre eles está o Misgurnus anguillicaudatus, este peixe tem também a capacidade invulgar de volatilizar a amónia. Uma dos métodos de tolerar níveis elevados de amónia é através da regulação do metabolismo da glutamina. As reacções de catabólise e anabólise deste metabolismo são catalizadas pela glutaminase (Gase), glutamina sintetase (GSase) e glutamato deshidrogenase (GDH) e permitem o consumo ou síntese de amónia. Estas reacções metabólicas poderão estar à volatilização da amónia no intestino do Misgurnus anguillicaudatus. O objectivo deste trabalho consistia em caracterizar a expressão dos três genes e do seu possível envolvimento na tolerância e volatilização da amónia. A expressão genética foi analisada no decurso de duas experiências. Numa primeira vários tecidos (cérebro, guelra, rim, fígado, e três partes do intestino), de animais controlo, seriam analisados de forma a determinar uma linha base da expressão dos genes. Na segunda experiência os níveis de expressão genética, para os três genes, em duas porções do intestino (intestino anterior e posterior), de animais expostos a elevados níveis de amónia ambiental, seriam quantificados ao longo do tempo para poder determinar possíveis alterações na expressão. Adicionalmente conduzir‐se‐ia a análise das sequências genéticas e comparação filogenética das sequências obtidas no decurso do trabalho. Os resultados da sequenciação mostraram elevada identidade entre as sequências do Misgurnus anguillicaudatus e os seus homólogos noutras espécies, mas na maior parte dos casos também havia distanciação evolutiva entre as sequências das espécies. Os resultados da expressão genética nos vários tecidos mostraram níveis significativamente elevados para a expressão da Gase e GSase no cérebro, estando de acordo com a importância destas enzimas na reciclagem do glutamato e na eliminação de amónia neste órgão. A expressão dos genes no intestino em resposta à exposição à amónia ao longo do tempo n mostraram alterações significativas, isto sugere que o metabolismo da glutamina não é importante para o processo de volatilização ou que regulação não se dá ao nível da transcrição. ABSTRACT: Many fishes are able to cope with high levels of environmental ammonia; among them is the oriental weatherloach (Misgurnus anguillicaudatus), which also has the unusual ability to volatilize ammonia. One proposed method of ammonia tolerance in this species is through the regulation of the glutamine metabolism. Glutaminase (Gase), glutamine synthetase (GSase) and glutamate dehydrogenase (GDH) catalyze reaction in which catabolysis and anabolysis lead to the release or consumption of ammonia, and can possibly be associated with ammonia volatilization in the gut of the weatherloach. This work intended to characterize the expression of these three genes and their possible involvement in ammonia tolerance and ammonia volatilization in the gut of the weatherloach. In order to determine the gene expression levels two experiments were analyzed separately. In one, several tissue samples (brain, gill, liver, kidney and three portions of gut) from control animals would be analyzed in order to determine baseline expression levels of the three genes. In the second experiment gene expression levels for the three genes in two sections of the gut (foregut and hindgut) from animals that had been exposed to high environmental ammonia over time would be analyzed in order to determine possible changes in gene expression. Additionally gene sequence analysis and phylogenic comparison would be carried out on the gene sequences obtained during the work. Sequencing results showed high identity between weatherloach and their homologues in other species for each gene, but in most cases with significant evolutionary branching from other species. The results for gene expression in several tissues showed significantly elevated levels of gene expression for Gase and GSase in the brain, which correlates with the importance of these enzymes in glutamate recycling and ammonia detoxification in this organ. The results for gene expression in gut in response to ammonia exposure over time showed no significant changes suggesting that glutamine metabolism is not important in ammonia volatilization or that regulation is not at the transcriptional level

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.J.A.K. acknowledges financial support from an Australian Government Research Training Program Scholarship. B.E.C. acknowledges financial support from a Rod Rickards PhD Scholarship. Funding by the Australian Research Council, including a Laureate Fellowship to G.O., is gratefully acknowledged. D.G. acknowledges the support of the Minerva Foundation, and this research was made possible in part by the historic generosity of the Harold Perlman Family (D.G.)

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as “ladybug taint”. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system

Determining the structure of the bacterial voltage-gated sodium channel NaChBac embedded in liposomes by cryo electron tomography and subtomogram averaging

Voltage-gated sodium channels shape action potentials that propagate signals along cells. When the membrane potential reaches a certain threshold, the channels open and allow sodium ions to flow through the membrane depolarizing it, followed by the deactivation of the channels. Opening and closing of the channels is important for cellular signalling and regulates various physiological processes in muscles, heart and brain. Mechanistic insights into the voltage-gated channels are difficult to achieve as the proteins are typically extracted from membranes for structural analysis which results in the loss of the transmembrane potential that regulates their activity. Here, we report the structural analysis of a bacterial voltage-gated sodium channel, NaChBac, reconstituted in liposomes under an electrochemical gradient by cryo electron tomography and subtomogram averaging. We show that the small channel, most of the residues of which are embedded in the membrane, can be localized using a genetically fused GFP. GFP can aid the initial alignment to an average resulting in a correct structure, but does not help for the final refinement. At a moderate resolution of ˜16 Å the structure of NaChBac in an unrestricted membrane bilayer is 10% wider than the structure of the purified protein previously solved in nanodiscs, suggesting the potential movement of the peripheral voltage-sensing domains. Our study explores the limits of structural analysis of membrane proteins in membranes

Mechanistic investigations on polyprenyl transferases and multi-target antibiotic discovery

Terpenoid biosynthesis plays essential structural and functional roles in almost all life forms. Prenyl transferases, enzymes involved in the isoprenoid metabolism related to prenyl chains, transfer allylic prenyl groups to acceptor molecules. Head-to-tail condensations, usually of allylic diphosphates (isoprene units), are catalyzed by head-to-tail prenyl transferases (commonly referred to prenyl diphosphate synthase), and produce regular terpenes which are by far the most common isoprenoid compounds. Head-to-middle prenyl transferases have been reported recently and they catalyze the formation of branched products which belong to irregular terpenes. Based on the stereochemical outcome of the linear products, head-to-tail prenyl transferases can be categorized to two major classes, trans- and cis- prenyl tranferases. Trans- prenyl transferases are critical players in bacterial respiratory systems while cis-prenyl transferases are involved in the biosynthesis of peptidoglycan, essential in bacterial cell wall synthesis. Thus, both classes of head-to-tail prenyl transferases are druggable targets in antibiotic development. In Chapter 2, I discuss the new series of lipophilic bisphosphonates that inhibit the growth of various bacteria and target multiple prenyl transferases. To determine and verify the enzyme targets of antibiotic leads, enzyme inhibition assays and bacterial rescue assays were performed. In Chapter 3, I discuss the antimicrobial agents phenylthiazoles which target bacterial UPPS, YubB (undecaprenyl diphosphate phosphatase (UPPP)) deducted from transposon mutagenesis. Putative inner membrane family proteins YubA and YubD are annotated as transporters and may also be targets because the compounds collapsed the proton motive force in membrane vesicles. Cis-prenyl transferase UPPS serves as a lipid carrier in cell wall peptidoglycan synthesis and catalyzes consecutive condensations of isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) to produce C55-PP. The UPPS product has the specific chain length essential for the biological function and an intriguing question is how UPPS regulates the product chain length. Chapter 4 is focused on chain length regulation mechanism of UPPS and its interaction with bacterial membrane. Head-to-middle prenyl transferases catalyze the formation of branched terpenoid products, which are reported more recently. It was found that they are highly structurally similar to the cis class of head-to-tail prenyl transferases. In Chapter 5, I discuss the first X-ray crystal structure of head-to-middle monoterpene synthase, lavandulyl pyrophosphate synthase (LPPS). Irregular prenyl transferase LPPS catalyzes two DMAPP molecules and form LPP, a precursor of the fragrances (R)-lavandulol and (R)-lavandulyl acetate. The active sites of LPPS were examined and compared with cis- head-to-tail prenyl transferase UPPS, and site-directed mutagenesis study was conducted. We proposed the first structure-based mechanism of action of this unusual prenyl synthase. In Chapter 6, a homolog of LPP, isosesquilavanduly diphosphate (ILPP), and its specific enzyme ILPPS were studied, and they are involved in the biosynthesis of the merochlorin class of antibiotics found in Streptomyces sp. strain CNH-189. We determined the crystal structure of ILPPS, substrate binding, substrate specificity, product length regulation, structure-based mechanism and the results were surprising and gave an unexpected perspective into catalysis by prenyl transferases. Based on ligand-bound crystal structure, the large, hydrophobic side chain of one substrate does not occupy a central hydrophobic channel. Instead, it occupies a surface pocket approximately 90° to the chain axis (hydrophobic tunnel) in other enzymes with cis- prenyl transferase fold. Interestingly, the proton abstraction is achieved with a diphosphate-Asn- Ser relay, which is shared in head-to-middle and cis- head-to-tail prenyl transferases

Development of dopamine and serotonin agonist radioligands for PET studies

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