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Supplementary Figure 1 from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

Supplementary Figure 2 from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

Supplementary Figure 3 from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

Supplementary Legends from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

Gluconate is a multidentate ligand and its complexation with actinides has received increasing attention because of its existence in high level nuclear wastes as well as in nuclear waste repositories. In this work, the complexation of gluconate with Th(IV) was studied in deuterated water (D2O) by pD titrations and nuclear magnetic resonance (NMR) spectroscopy. In the pCD range 2.0–4.6, gluconate (GD4–) forms two 1:1 complexes with Th(IV), Th(GD3)2+ and Th(GD2)+. Their stability constants were determined to be log β(D)101(−1) = 1.04 ± 0.12 for Th4+ + GD4– = Th(GD3)3+ + D+ and log β(D)101(−2) = −(1.31 ± 0.09) for Th4+ + GD4– = Th(GD2)+ + 2D+ at I = 1.0 mol·L–1 NaClO4 and t = 22 °C. The coordination modes of these two complexes were also analyzed. In both complexes, the tridentate chelation forms through the binding of Th(IV) to one oxygen from the carboxylate group and two oxygens from α- and γ-hydroxyl groups. The difference is that in Th(GD2)+, both α- and γ-hydroxyl groups deprotonate, and in Th(GD3)2+, ...

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a “swinging gate” allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance., Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance, but its role is unclear. A 3D model of a complex between leiomodin and the actin pointed end reveals a mechanism of regulating the length of thin filaments in striated muscle.

Made available in DSpace on 2018-12-11T16:52:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 The full use of biomass in future biorefineries has stimulated studies on utilization of lignin from agricultural crops, such as coffee husk, a major residue from coffee processing. This study focuses on characterizing the lignin obtained from coffee husk and its further wet oxidation products as a function of alkali loading, temperature and residence time. The lignin fraction after diluted acid and alkali pretreatments is composed primarily of p-hydroxylphenyl units (≥49%), with fewer guaiacyl and syringyl units. Linkages appear to be mainly β-O-4 ether linkages. Thermal degradation of pretreated lignin during wet oxidation occurred in two stages. Carboxylic acids were the main degradation product. Due to the condensed structure of this lignin, relatively low yields of aromatic aldehydes were achieved, except with temperatures over 210 °C, 5 min residence time and 11.7 wt% NaOH. Optimization of the pretreatment and oxidation parameters are important to maximizing yield of high-value bioproducts from lignin. Biotechnology Department Engineering School of Lorena University of São Paulo Bioproducts Sciences and Engineering Laboratory Washington State University, Tri-Cities Center for NMR Spectroscopy Washington State University William R. Wiley Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory Voiland School of Chemical and Bioengineering and Biological Systems Engineering Washington State University Co-author currently works in Bioenergy Research Institute UNESP Co-author currently works in Bioenergy Research Institute UNESP

In this work, we demonstrated the successful synthesis of porous organic polymers via a ball-milling procedure. Several readily available benzene derivatives were selected to be polymerized through a Friedel–Crafts reaction with FeCl3 as the Lewis acid catalyst and formaldehyde dimethyl acetal as the crosslinker. All the mechano-synthesized porous organic polymers (MPOPs) are not soluble in common organic solvents, and the calculated surface area was over 500 m2 g−1 when biphenyl was used as the monomer. One of the advantages of applying ball-milling in targeted polymer synthesis is bypassing the use of large quantity of hazardous chlorinated solvents, which are commonly used in traditional Friedel–Crafts reactions. Considering the aromatic skeleton and hydrophobic nature of these polymers, their performance in p-nitrophenol (PNP) adsorption from water was investigated. The quantification was carried out on an Ionics 3Q 320 LC-MS/MS system with 4-nitrocatechol (PNC) as an internal standard. MPOP-1 and MPOP-3 showed the maximum adsorption capacity of 133.10 and 155.51 mg g−1 for PNP, respectively. Their adsorption kinetics were studied and both adsorption isotherms were well delineated with a pseudo-second-order equation, indicating the availability of strong adsorption sites in both MPOPs for interacting with PNP.

This paper reports a study of the chemical composition of the water-soluble (WS) fraction obtained by cold water precipitation of two commercial wood pyrolysis oils (BTG and Amaron). The fraction studied accounts for between 50.3 and 51.3 wt % of the oils. With the most common analytical techniques used today for the characterization of this fraction (KF titration, GC–MS, hydrolyzable sugars, and total carbohydrates), it is possible to quantify only between 45 and 50 wt % of the fraction. Our results confirm that most of the total carbohydrates (hydrolyzable sugars and nonhydrolyzable) are soluble in water. The ion chromatography hydrolysis method showed that between 11.6 and 17.3 wt % of these oils were hydrolyzable sugars. A small quantity of phenols detectable by GC–MS (between 2.5 and 3.9 wt %) were identified. It is postulated that the unknown high molecular weight fraction (30–55 wt %) is formed by highly dehydrated sugars rich in carbonyl groups and WS phenols. The overall content of carbonyl, carb...

The precise arrangement of the N-terminal actin- and tropomyosin-binding sites within leiomodin-2 is determined. A novel model is given of the roles of leiomodin-2 at thin filament pointed ends., Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2–knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43–90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124–201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly­merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

Quantification of functional groups (carbonyl, carboxyl, hydroxyl, phenolics) in biomass-derived pyrolysis oils is crucial to advance our understanding of bio-oil compositional changes during production, storage, aging, and upgrading. Traditionally, most of the methods reported in the literature on this subject are based on titration. There are very few studies on the use of spectroscopic techniques for the quantification of functional groups in bio-oils. The distribution of functional groups between the volatile and the heavy fraction is also very poorly understood. The content of functional groups in the volatile fraction estimated by GC/MS was compared with their content in the total oil determined by titration and 31P NMR. The carbonyl groups are almost equally distributed between the volatile and the oligomeric fractions. The content of total phenols varies between 1.6 and 3.1 mmol/g. It is important to note that between 85 and 95% of the phenols in bio-oil are in the form of oligomers. The content o...

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

The ability of winter wheat (Triticum aestivum L.) plants to develop freezing tolerance through cold acclimation is a complex rait that responds to many environmental cues including day length and temperature. A large part of the freezing tolerance is conditioned by the C-repeat binding factor (CBF) gene regulon. We investigated whether the level of freezing tolerance of 12 winter wheat lines varied throughout the day and night in plants grown under a constant low temperature and a 12-hour photoperiod. Freezing tolerance was significantly greater (P

Most current lignin content analysis methods require dissociation/degradation and/or preconditioning steps. A simple yet reliable method that can be used to analyze content of various lignocellulosic biomasses without destroying their native structure is needed. In this research effort, an improved nondegradative method for determining the content of lignin in native biomass was established. By extending the scope of the solid state 13C CP/MAS NMR technique, via homogeneous mixing of an internal standard (TMSP) with varying lignin contents, a standard curve was generated for calculating the lignin content of various biomasses without dissociation or degradation. The results showed good accuracy in the calculation of lignin content. Compared to chemical degradative methods, such as the acetyl bromide method, the relative difference was less than 5.00%. This method is applicable for estimating lignin content in certain samples (e.g., acid insoluble lignin) that are difficult to analyze accurately with chemi...

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from 1H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform–paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple 1H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co2+-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH151–256, the heme sensing domain of Bradyrhizobium japonicum FixL. Copyright © 2013 John Wiley & Sons, Ltd.

Harvested forest residues are usually considered a fire hazards and used as “hog-fuel” which results in air pollution. In this study, the biorefinery lignin stream obtained after wet explosion pretreatment and enzymatic hydrolysis of forestry residues of Douglas Fir (FS-10) was characterized and further wet oxidized under alkaline conditions. The studies indicated that at 10% solids, 11.7 wt% alkali and 15 min residence time, maximum yields were obtained for glucose (12.9 wt%), vanillin (0.4 wt%) at 230 °C; formic acid (11.6 wt%) at 250 °C; acetic acid (10.7 wt%), hydroxybenzaldehyde (0.2 wt%), syringaldehyde (0.13 wt%) at 280 °C; and lactic acid (12.4 wt%) at 300 °C. FTIR analysis of the solid residue after wet oxidation showed that the aromatic skeletal vibrations relating to lignin compounds increased with temperature indicating that higher severity could result in increased lignin oxidation products. The results obtained, as part of the study, is significant for understanding and optimizing processes for producing high-value bioproducts from forestry residues.

The development of some familial dilated cardiomyopathies (DCM ) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Whereas the biological consequence of mutations affecting protein folding, stability and levels of intracellular expression is more apparent, understanding of harmful effects of mutations interfering with yet uncharacterized protein-protein interactions might be elusive. We used nuclear magnetic resonance spectroscopy to precisely localize the region of striated muscle α-tropomyosin that interacts with leiomodin-2 (Lmod2), a member of tropomodulin family of actin-binding proteins. The K15N mutation known to be associated with familial DCM is located within the newly identified Lmod2 binding site of tropomyosin. We studied the effect of this mutation on binding Lmod2 and Tmod1, cardiac isoforms responsible for correct lengths of the thin filaments. The mutation reduced binding affinity for both Lmod2 and Tmod1, however the decrease was more pronounced for binding Lmod2. The reduced affinity and the uneven effect of the K15N mutation on TM binding to Lmod2 and Tmod1 provide a molecular rationale for the development of familial DCM.

Non-invasive methods for measuring lipid content in live microalgal cultures are critically needed for algal biofuel research and development. A non-destructive method requiring minimal sample preparation was developed utilizing liquid state ¹H NMR for quantifying triacylglycerides (TAGs) in live algae cultures. ¹H NMR and ¹H HR-MAS spectra of live algae cells show outstanding correlation with published chemical shifts for TAGs. ¹H NMR spectra of Chlamydomonas reinhardtii whole cells and isolated lipid bodies were compared with a standard oleic TAG ¹H spectrum, demonstrating that only lipid body TAGs were observed. A “model” TAG was derived, providing a proton count and molecular weight for conversion of TAG ¹H NMR integrals to volumetric TAG or fatty acid methyl ester (FAME) equivalent concentrations, which were correlated to FAME concentrations by gas chromatography (FAME-GC) at several time points. A customized NMR flow cell was subsequently constructed, allowing real-time, continuous measurements of multiple cultures.

In weakly acidic aqueous solutions, uranyl is able to slowly photolyze gluconic acid under ambient laboratory light (fluorescence lamps). The photolysis follows “complex formation”, resulting in the decarboxylation of gluconic acid (CH2OH(CHOH)4COOH) to form D-arabinose ((CHOH)4OCH2). A possible pathway is proposed to be the sensitized photo-oxidation, which can be described as U(VI)·CH2OH(CHOH)4COOH → U(V) + CH2OH(CHOH)3ĊHOH + CO2 + H+ and U(V) + CH2OH(CHOH)3ĊHOH + H+ → U(VI) + (CHOH)4OCH2 + H2. Also, characterization of the precipitate observed in the photolytic samples demonstrates the presence of tetravalent U, which we believe is generated by concurrent disproportionation of intermediate pentavalent U.

Within the pC(H) range of 2.5 to 4.2, gluconate forms three uranyl complexes UO(2)(GH(4))(+), UO(2)(GH(3))(aq), and UO(2)(GH(3))(GH(4))(-), through the following reactions: (1) UO(2)(2+) + GH(4)(-) = UO(2)(GH(4))(+), (2) UO(2)(2+) + GH(4)(-) = UO(2)(GH(3))(aq) + H(+), and (3) UO(2)(2+) + 2GH(4)(-) = UO(2)(GH(3))(GH(4))(-) + H(+). Complexes were inferred from potentiometric, calorimetric, NMR, and EXAFS studies. Correspondingly, the stability constants and enthalpies were determined to be log beta(1) = 2.2 +/- 0.3 and DeltaH(1) = 7.5 +/- 1.3 kJ mol(-1) for reaction (1), log beta(2) = -(0.38 +/- 0.05) and DeltaH(2) = 15.4 +/- 0.3 kJ mol(-1) for reaction (2), and log beta(3) = 1.3 +/- 0.2 and DeltaH(3) = 14.6 +/- 0.3 kJ mol(-1) for reaction (3), at I = 1.0 M NaClO(4) and t = 25 degrees C. The UO(2)(GH(4))(+) complex forms through the bidentate carboxylate binding to U(VI). In the UO(2)(GH(3))(aq) complex, hydroxyl-deprotonated gluconate (GH(3)(2-)) coordinates to U(VI) through the five-membered ring chelation. For the UO(2)(GH(3))(GH(4))(-) complex, multiple coordination modes are suggested. These results are discussed in the context of trivalent and pentavalent actinide complexation by gluconate.

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.

A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with kenz approximately 10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at approximately 100 microg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this "knockout" had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

The isolation and structure elucidation of flutimide (1), an inhibitor of flutranscription endonuclease from Delitschia confertaspora, a new species, is reported. The novel natural product is characterized by N-hydroxyimide and exocyclic enamine functionalities.

The antimicrobial agent pramanicin ( 1 ), and a related fatty acid ( 6 ), were isolated from a corn-based solid or a lactose-containing liquid fermentation of a sterile fungus found growing in grass. The structures of these compounds were determined by a variety of spectral means including UV, IR, and NMR spectroscopy, as well as mass spectrometry. A number of chemical derivatives are also presented here. Pramanicin represents a new class of antimicrobial agents containing a highly functionalized head group and functionalized fatty side chain

Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble.

Radical SAM (S-adenosyl-L-methionine), cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes have been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea. PhpK catalyzes the transfer of a methyl group from methylcobalamin to 2-acetylamino-4-hydroxyphosphinylbutanoate (N-acetyldemethylphosphinothricin or NAcDMPT) to form 2-acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphosphinothricin or NAcPT). This transformation gives rise to the only carbon-phosphorus-carbon linkage known to occur in Nature.

The taccalonolides are a unique class of microtubule stabilizers that do not bind directly to tubulin. Three new taccalonolides, Z, AA, and AB, along with two known compounds, taccalonolides R and T, were isolated from Tacca chantrieri and Tacca integrifolia. Taccalonolide structures were determined by 1D and 2D NMR methods. The biological activities of the new taccalonolides, as well as taccalonolides A, B, E, N, R, and T, were evaluated. All nine taccalonolides display microtubule stabilizing activity, but profound differences in antiproliferative potencies were noted, with IC(50) values ranging from the low nanomolar range for taccalonolide AA (32 nM) to the low micromolar range for taccalonolide R (13 μM). These studies demonstrate that diverse taccalonolides possess microtubule stabilizing properties and that significant structure-activity relationships exist. In vivo antitumor evaluations of taccalonolides A, E, and N show that each of these molecules has in vivo antitumor activity.

The isolation and characterization of calbistrin A [1] were complicated by the compound's instability. The instability was caused by light, which led to a complex degradation of 1. The nature of the light instability was elucidated by forming the dimethyl acetal derivative 2, which isomerized upon exposure to light, leading to an equilibrium mixture of 2 and 3. Alkaline conditions also caused decomposition of 1, leading to the formation of4

The isolation and structure elucidation of three members of a novel family of amino acid containing alkyl citrates, viridiofungins A (1), B (2) and C (3), are described. They are potent, broad spectrum antifungal agents and in vitro inhibitors of squalene synthase.

A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.

The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the βIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the βIII isotype of tubulin was generated to evaluate the effect of βIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the βIII-tubulin–overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the βIII-tubulin–overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential. [Cancer Res 2008;68(21):8881–8]

An enantio-specific polyphenol oxidase (PPO) was purified ≈1,700-fold to apparent homogeneity from the creosote bush ( Larrea tridentata ), and its encoding gene was cloned. The posttranslationally processed PPO (≈43 kDa) has a central role in the biosynthesis of the creosote bush 8–8′ linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3′-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (–)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.

In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.

Regiospecifically labeled geranyl diphosphates ((2E,6E)-[1,1,8,8,8-(2)H(5)]- and (2E,6Z)-[1,1,9,9,9-(2)H(5)]-GDP) and D(2)O incorporation, in concert with NMR spectrometry, were employed to demonstrate a unique intramolecular syn-facial protonation-cyclization mechanism of action of 1,8-cineole synthase.

Conversion of biomass derived lignin to liquid fuels has the promising potential to significantly improve carbon utilization and economic competitiveness of biomass refineries. In this study, an aqueous phase catalytic process was developed to selectively depolymerize the lignin polymeric framework and remove oxygen via hydrodeoxygenation (HDO) reactions. Efficient methods (ethanol and dilute alkali extraction) for selectively producing reactive lignin oligomers with high yields from corn stover were established. Characteristic structural features of the technical lignins employed for hydrocarbon production were elucidated with the aid of advanced analytical techniques, such as 2D HSQC NMR spectroscopy and gel permeation chromatography (GPC). Combinations of noble metal catalysts in the presence of various solid acid zeolites were tested for HDO activity of the oligomeric technical lignins predominantly containing 8–O–4′ inter-unit linkages. Results showed 35%–60% conversion of lignin with 65%–70% product selectivity for aromatic hydrocarbons (e.g. toluene) under various HDO conditions in the presence of noble metals (Ru, Rh and Pt) over Al2O3 (or C) supports and solid acid zeolites (e.g., NH4+ Z-Y 57277-14-1) catalyst matrices.

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.