Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli

Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.     

New function of the amino group of thiamine diphosphate in thiamine catalysis

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4′-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase—4′-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4′-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH²-methylated ThDP exhibits dihydroxyethyl-4′-methylamino-ThDP-synthase activity. Thus, the 4′-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.     

The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.     

Induced absorption band of holotransketolase and its interpretation

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1"-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.     

X-ray crystallographic snapshots of reaction intermediates in pyruvate oxidase and transketolase illustrate common themes in thiamin catalysis

Thiamin diphosphate (ThDP)-dependent enzymes play pivotal roles in intermediary metabolism of virtually all organisms. Although extensive mechanistic work on cofactor models and various enzymes has served as a guide to understand general principles of catalysis, high-resolution structural information of reaction intermediates along the catalytic pathway was scarcely available until recently. Here, we review cryocrystallographic studies on the prototypical ThDP enzymes pyruvate oxidase and transketolase, which provided exciting insights into the chemical nature and structural features of several key intermediates and into the stereochemical course of substrate processing. The structures revealed a conserved (S)-configuration at the C2alpha stereocenter of the initially formed tetrahedral intermediate in the different enzymes with the scissile C2alpha–C2beta bond being directed perpendicular to the aromatic ring plane of the thiazolium portion of ThDP confirming the proposed maximum overlap mechanism. Elimination of the respective leaving groups (carbon dioxide, sugar phosphates) appears to be driven – amongst other factors such as stereoelectronic control – by strain relief as the C2–C2alpha bond, which connects C2 of ThDP with the carbonyl of the substrate, substantially deviates from planarity and relaxes to an in-plane conformation only after bond fission to give an enamine-type intermediate with considerable delocalization of the free electron pair onto the thiazolium ring. Except for the apparent flexibility of the cofactor itself, no major structural rearrangements are detectable indicating that the enzyme active centers are poised for catalysis. The structures also provide the basis for understanding the origins of substrate and reaction specificity.     

C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models

Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects

Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.     

Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.     

A versatile conformational switch regulates reactivity in human branched-chain alpha-ketoacid dehydrogenase

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.     

Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase

The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP <--> TK...ThDP <--> TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.     

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.     

Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and beta-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Investigation of the donor and acceptor range for chiral carboligation catalyzed by the E1 component of the 2-oxoglutarate dehydrogenase complex

The potential of thiamin diphosphate (ThDP)-dependent enzymes to catalyze CC bond forming (carboligase) reactions with high enantiomeric excess has been recognized for many years. Here we report the application of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex in the synthesis of chiral compounds with multiple functional groups in good yield and high enantiomeric excess, by varying both the donor substrate (different 2-oxo acids) and the acceptor substrate (glyoxylate, ethyl glyoxylate and methyl glyoxal). Major findings include the demonstration that the enzyme can accept 2-oxovalerate and 2-oxoisovalerate in addition to its natural substrate 2-oxoglutarate, and that the tested acceptors are also acceptable in the carboligation reaction, thereby very much expanding the repertory of the enzyme in chiral synthesis.     

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.     

Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.     

Steric and electronic properties of the cofactor's amino group control the lifetime of the central carbanion/enamine intermediate in transketolase

Transketolase (TK), a thiamin diphosphate (ThDP) dependent enzyme, catalyzes the reversible transfer of a two-carbon unit from keto- to aldo-substrates. Dihydroxyethylthiamin diphosphate (DHEThDP), formed as a result of cleavage of the donor substrate, serves as an intermediate of the TK reaction. TK from the yeast Saccharomyces cerevisiae is unique among thiamin enzymes displaying enzymatic activity after reconstitution with a methylated analogue of the native cofactor, 4′-methylamino-ThDP. The reconstitution of the apoenzyme with both ThDP and the methylated analogue can be analyzed by near UV circular dichroism. It was demonstrated that in the native holoenzyme and in the complex of TK with 4′-methylamino-ThDP the formation of the dihydroxyethyl-based carbanion/enamine took place with comparable rate constants, whereas the protonation of the reactive species was much faster in the complex with the analogue. The enzymatic activity of the enzyme reconstituted with 4′-methylamino-ThDP was 10fold higher in the ferricyanide assay. We suggest that a methylation of the 4′-amino group of ThDP impairs the resonance stabilization of the carbanion/enamine intermediate both sterically and electronically, thus allowing either a faster protonation or oxidation reaction by ferricyanide. The formation of the optically active DHE-4′-methylamino-ThDP was monitored by near UV circular dichroism spectra and corroborated by ¹H NMR analysis. The protonated form of the intermediate DHE-4′-methylamino-ThDP was released from the active sites of TK and accumulated in the medium on preparative scale.     

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.     

Chemotaxonomic relevance of cardenolides in Urginea fugax

In a chemotaxonomic approach the investigation of a methanolic extract of bulbs of Urginea fugax (MORIS) STEINH. resulted in the detection of several cardenolides. The structure of a novel compound, named fugaxin (1), was established as 12alpha,14beta-dihydroxy-2alpha,3beta-(tetrahydro-3',5'-dihydroxy-4'-methoxy-6'-methyl-2H-pyran-2',4'-diylbisoxy)-card-4,20-dienolide by extensive NMR spectroscopic studies including 2D-NMR techniques (COSY, HSQC, HMQC) and selective 1D experiments (NOE, TOCSY) as well as HR-ESI-MS. The chemotaxonomic relevance of the occurrence of cardenolides in the genus Urginea is discussed.     

Subproteomic analysis of the cellular proteins associated with the 3' untranslated region of the hepatitis C virus genome in human liver cells

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication and protein translation by interacting with the viral and host components. To examine host proteins interacting with the HCV 3'UTR, biotinylated 3'(+)UTR, and its reverse complementary 5'(-)UTR were used in RNA pull-down assay. Cellular proteins from Huh7 cells pulled down by biotinylated RNAs were identified by 2DE/MALDI-TOF MS and 1DE/LC/MS methods. Totally, 10 proteins could be identified from both methods, among which six bound specifically to the 3'(+)UTR, three proteins to the 5'(-)UTR only, and one protein bound to both. Three identified proteins (PCBP2, G3BP1, and DDX1) were selected for further investigation into their possible roles on the HCV replication. Differently regulating effects on HCV replication by siRNA-mediated silencing of these proteins were observed, indicating a complex role of 3'UTR binding proteins on HCV replication.