NMR-spektroskopische Untersuchungen an katalytischer RNA : ein Spleißosom basiertes lariatformendes Ribozym ; die Konformation der 2'-Hydroxylgruppe in RNA und deren Rolle in der Strukturstabilität

In Eukaryonten findet der Prozess des Spleißens im Spleißosom, einer sich permanent reorganisierenden, molekularen Maschine, statt. In diesem Prozess werden die kodierenden Sequenzen, die Exons, von den nicht kodierenden Sequenzen, den Introns, getrennt und zusammengefügt. Durch die permanente Reorganisation des Spleißosoms war es bisher nicht möglich, die dreidimensionale Struktur der unterschiedlichen Komplexe in einer hohen Auflösung zu bestimmen. Tuschl et al. haben ein Ribozym entwickelt. Dieses bildet über den Angriff einer 2'-Hydroxylgruppe eines Adenosins auf das Phosphatrückgrat eines Guanosins ein Lariat. Diese Reaktion ist vergleichbar dem ersten Schritt der Spleißreaktion. In dieser Arbeit wurde das Ribozym vor der Ausbildung der Lariatstruktur charakterisiert. Eine Voraussetzung zur Strukturaufklärung mit NMR ist die Zuordnung der Resonanzen. Die Zuordnung zeigte, dass ein Teil der linearen Form des Ribozyms in zwei Konformationen vorliegt. Hier findet ein langsamer konformationeller Austausch statt, der die Resonanzüberlagerung erhöht und damit die Zuordnung der Signale erschwert. Aus diesem Grund wurden unterschiedliche Isotopen markierte Proben für die Zuordnung verwendet. Zur Verifizierung der Sekundärstruktur wurden neben NMR-Experimenten Mutationsstudien am Ribozym eingesetzt. Das Ribozym bildet zwischen g1-c12 und g17-c27 eine Helix. Die Helix weist neben kanonischen Basenpaarungen auch nicht kanonische Basenpaarungen auf. Die Adenosine am Anfang der Helix interagieren mit Nukleotiden in 3'-Position des branchpoint Adenosins. Dadurch befindet sich das branchpoint Adenosin a48 nahe dem g1. Zwischen diesen beiden Nukleotiden findet die Umesterung statt, in der das Lariat gebildet wird. Nach der Lariatbildung sind in den NMR Spektren nur noch die Resonanzen der kurzen Helices P1 und P2 zu beobachten. Die anderen Resonanzen unterliegen einem konformationellen Austausch. Die Struktur des Ribozyms verändert sich also nach der Lariatbildung beträchtlich. In der Spleißreaktion, aber auch in anderen katalytischen Prozessen besitzt die 2'-Hydroxylgruppe der RNA eine essentielle Funktion. Auch die wesentlichen strukturellen Unterschiede zwischen der RNA und DNA sind auf die 2'-Hydroxylgruppe zurückzuführen. Bislang liegen jedoch nur moleküldynamische Berechnungen und NMR-spektroskopische Untersuchungen an einzelnen Nukleotiden vor, die sich mit der Konformation der 2'-Hydroxylgruppe beschäftigen. In dieser Arbeit wurden erstmals die Konformationen der 2'-Hydroxylgruppen einer dreißig Nukleotide umfassenden RNA, der TAR-RNA, mit Hilfe von 3J-Kopplungen und NOEs bei niedrigen Temperaturen in Lösung ermittelt. Die Konformationsanalyse ergibt, dass die 2'-Hydroxylgruppen der unteren Helix der TAR-RNA in einem konformationellen Gleichgewicht zwischen der O3'- und der Basendomäne vorliegen. In beiden Orientierungen können sich die 2'-Hydroxylgruppen am Aufbau eines Netzwerks aus Wassermolekülen beteiligen, in dem zwei Wassermoleküle die große Furche der RNA Helix überspannen. Durch den Wechsel der 2'-Hydroxylgruppen zwischen der O3'- und Basen-Domäne unterliegt das Netzwerk einer stetigen Reorganisation.Eukaryotic splicing, whereby the coding exon sequences are separeted from the noncoding introns of pre-mRNA, is mediated through the spliceosome. Tuschl and coworkers designed a ribozyme which is based on the U2- und U6-snRNAs of the active complex of the spliceosome. As in the first step of splicing, it forms a lariat structure. In this transesterfication reaction a 2’-hydroxyl group of an adenosine attacks the phosphate backbone of a guanosine. The leaving group in the ribozyme is a pyrophosphate rather than the 5’-exon in the spliceosome. Due to the permanent reorganization of the spliceosome, the structure of the active complex is unknown. For this reason the lariat forming ribozyme can provide insight into the structural and functional features of the splicing reaction. Resonance Assignment is crucial for structure determination by NMR. Half of the linear ribozyme, including the short helix P2, is subject to a conformational exchange on a millisecond timescale. The exchange impedes the assignment because these nucleotides exhibit two different sets of resonances leading to resonance overlap. For the investiagtion of the lariat forming ribozyme a number of samples with differential isotopic labelling together with NMR experiments based on the NOE with filtering and editing techniques were found to be most suitable. The linear ribozyme forms a helix between nucleotides g1-c12 and g17-c27 as shown by NMR experiments and mutational studies. In this helix, canonical as well as non-canonical basepairs are included. Nucleotide g49, next to the branchpoint adenosine a48, is in close proximity to guanosine g1. This juxtaposes the 2’-hydroxy group of a48 next to the triphosphate at the 5’-end of the ribozyme and allows the transesterfication reaction. After lariat formation, the NMR spectra only show the resonances of the short helices P1 and P2. The other resonances are subject to a conformational exchange in the micro- to millisecond timescale. Upon lariat formation various structural changes occur within the ribozyme. The 2’-hydroxyl group of RNA plays an important role in, not only splicing, but numerous other catalytic processes. It is also responsible for the structural and functional differences between RNA and DNA. The conformation of this functional group has only been studied by molecular dynamic simulations and NMR of single nucleotides. In this work a conformational analysis of the 2’-hydroxyl group for the 30-mer HIV-2 transactivation response element (TAR) RNA was performed with 3J-couplings and NOEs at low temperature in solution. The analysis shows that the 2’-hydroxyl groups of the lower stem are in conformational exchange between the O3’ domain and the base domain. The 2’-hydroxyl proton interacts electrostatically in the O3’ domain wih the oxygen atom of the phosphate backbone, in the base domain with the O2 or N3 atom. The 2’-hydroxyl groups of paired nucleotides adopt two conformations. In the first, both groups are within the base domain. In the second one group shift to the O3’ domain. In both situations it enables a water network to form within the minor groove

Phenylacrylic acids addition to potato and sweet potato showed no impact on acrylamide concentration via oxa-Michael-addition during frying

Three phenolic acids, p-coumaric, ferulic and caffeic acid as well as cinnamic acid were added to raw potatoes and sweet potatoes before frying. A distinct mitigation of acrylamide was not detected. Fried samples were analysed for postulated adducts of a direct reaction between acrylamide and these phenolic acids using LC-MS. In a model system with pure compounds (phenylacrylic acid and acrylamide) heated on 10% hydrated silica gel one specific adduct (respective m/z for M ​+ ​H+) was formed in each reaction. MS/MS-data suggested an oxa-Michael formation of 3-amino-3-oxopropyl-phenylacrylates, which was confirmed by de novo syntheses along an SN2 substitution of 3-chloropropanamide. Exemplarily, the structure of the ester was confirmed for p-coumaric acid by NMR-data. Standard addition revealed that 3-amino-(3-oxopropyl-phenyl)-acrylates occurred neither in fried potato nor in sweet potato, while a formation was shown in phenylacrylic acid plus acrylamide supplemented potatoes and sweet potatoes

Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis. © 2020, The Author(s)

Rational reprogramming of the sesquiterpene synthase BcBOT2 yields new terpenes with presilphiperfolane skeleton

Computer-aided rational design of the substrate binding pocket of sesquiterpene synthases BcBOT2 from Botrytis cinerea yielded FPP cyclization products with presilphiperfolane backbone other than the naturally formed sesquiterpene presilphiperfolan-8β-ol. Particularly, amino acids W118 and F138 were found to strongly control the stability and conformation of key cationic intermediates. The W118Q variant forms only presilphiperfolan-9β-ol, whereas the exchange of amino acids at position 138, such as F138V, has a fundamental effect on the course of the cationic cascade. Here, the 1,3-hydride shift en route to presilphiperfolan-8β-ol is suppressed and substituted by a so far unknown 1,2-hydride shift that leads to presilphiperfol-1-ene and presilphiperfolan-1α-ol along with β-caryophyllene and the so far unknown caryophyllene-8β-ol

Determination of the structural integrity and stability of polysialic acid during alkaline and thermal treatment

Polysialic acid (polySia) is a linear homopolymer of varying chain lengths that exists mostly on the outer cell membrane surface of certain bacteria, such as Escherichia coli (E. coli) K1. PolySia, with an average degree of polymerization of 20 (polySia avDP20), possesses material properties that can be used for therapeutic applications to treat inflammatory neurodegenerative diseases. The fermentation of E. coli K1 enables the large-scale production of endogenous long-chain polySia (DP ≈ 130) (LC polySia), from which polySia avDP20 can be manufactured using thermal hydrolysis. To ensure adequate biopharmaceutical quality of the product, the removal of byproducts and contaminants, such as endotoxins, is essential. Recent studies have revealed that the long-term incubation in alkaline sodium hydroxide (NaOH) solutions reduces the endotoxin content down to 3 EU (endotoxin units) per mg, which is in the range of pharmaceutical applications. In this study, we analyzed interferences in the intramolecular structure of polySia caused by harsh NaOH treatment or thermal hydrolysis. Nuclear magnetic resonance (NMR) spectroscopy revealed that neither the incubation in an alkaline solution nor the thermal hydrolysis induced any chemical modification. In addition, HPLC analysis with a preceding 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization demonstrated that the alkaline treatment did not induce any hydrolytic effects to reduce the maximum polymer length and that the controlled thermal hydrolysis reduced the maximum chain length effectively, while costeffective incubation in alkaline solutions had no adverse effects on LC polySia. Therefore, both methods guarantee the production of high-purity, low-molecular-weight polySia without alterations in the structure, which is a prerequisite for the submission of a marketing authorization application as a medicinal product. However, a specific synthesis of low-molecular-weight polySia with defined chain lengths is only possible to a limited extent

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism

Conformational preferences of natural and C3-modified epothilones in aqueous solution

The conformational properties of the microtubule-stabilizing agent epothilone A ( 1a) and its 3-deoxy and 3-deoxy-2,3-didehydro derivatives 2 and 3 have been investigated in aqueous solution by a combination of NMR spectroscopic methods, Monte Carlo conformational searches, and NAMFIS calculations. The tubulin-bound conformation of epothilone A ( 1a), as previously proposed on the basis of solution NMR data, was found to represent a significant fraction of the ensemble of conformations present for the free ligands in aqueous solution

Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene

Cystobactamids 920-1 and 920-2: Assignment of the Constitution and Relative Configuration by Total Synthesis.

Total synthesis of cystobactamid 920-1 and its epimer has allowed an unambiguous assignment of the relative and absolute configuration of the natural product. A careful structural analysis of each isomer using both NMR and computational techniques also prompted a constitutional revision of the structures originally reported for cystobactamids 920-1 and 920-2, and has provided further insight into the unique conformational preferences of the cystobactamid famil