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The antibacterial and antiviral agent amicetin is a disaccharide nucleoside antibiotic featuring a unique α-(1→4)-glycoside bond between amosamine and amicetose, characteristic of a retaining glycosylation. In this study, two key steps for amosamine biosynthesis were investigated: the N- methyltransferase AmiH was demonstrated to be requisite for the dimethylation in amosamine, and the glycosyltransferase AmiG was shown to be necessary for amosaminylation. Biochemical and kinetic characterization of AmiG revealed for the first time the catalytic reversibility of a retaining glycosyltransferase involved in secondary metabolite biosynthesis. AmiG displayed substrate flexibility by utilizing five additional sugar nucleotides as surrogate donors. AmiG was also amenable to sugar and aglycon exchange reactions. This study indicates that AmiG is a potential catalyst for diversifying nucleoside antibiotics and paves the way for mechanistic studies of a natural-product retaining glycosyltransferase. [ABSTRACT FROM AUTHOR]

Parameter estimation in thermodynamic models has been of great interest in chemical engineering because of its complex nature, including nonlinearity, flat objective function in the neighborhood of the global optimum, badly scaled model, and nondifferential term(s) in the equations. In this article, a stochastic global optimization algorithm called integrated differential evolution (IDE) is introduced to solve the parameter estimation problems for modeling vapor–liquid equilibrium (VLE) data. In IDE, the choice of mutation strategy and associated parameters are adapted according to the learning experience from previous generations. The tabu list used in IDE can avoid revisiting the same area and avoid unnecessary function evaluations. A novel and effective stopping criterion based on the number of rejected points during the generation of a trial vector is tested and compared with other criteria. Furthermore, IDE uses a local optimizer after the global search to find the optimum accurately and efficiently. The performance of IDE for benchmark functions and VLE modeling is compared with that of other stochastic algorithms such as DE, DE with tabu list, particle swarm optimization, simulated annealing, and the deterministic algorithm Branch and Reduce Optimization Navigator (BARON). IDE is shown to be better than or comparable to these algorithms for parameter estimation in modeling VLE data. [ABSTRACT FROM AUTHOR]

A series of novel wholly aromatic sulfonated poly(arylene ether) copolymers containing sulfonic acid groups on the pendants SC-SPAE, i.e., side-chain-type sulfonated poly(arylene ether) were prepared by direct copolymerization of sulfonated monomer sodium 4-(4-(2,6-difluorobenzoyl)phenoxy) benzenesufonate (SDFBS), 4,4‘-dichlorodiphenyl sulfone (DCDPS), and 4,4‘-dihydroxyldiphenylether (DHDPE). The sulfonate degree (DS) of the copolymers was readily controlled by adjusting the feed ratio of SDFBS to DCDPS. Structures of the sulfonated copolymers were confirmed by FT-IR and NMR. Tough and flexible films of the wholly aromatic copolymers with good thermal and oxidative stability were obtained by solvent cast process in N,N-dimethylacetamide (DMAc) solution. The films of these side-chain-type copolymers show good dimensional stability, and their water uptake and swelling ratios at high temperature are lower than those of the main-chain-type sulfonated poly(arylene ether) with similar ion exchange capacity (IEC) value. Proton conductivities of the copolymers with high sulfonate degree (DS > 0.7) are higher than 10-2S/cm and increase gradually with increasing temperature. At 100 °C, the conductivities of SC-SPAE90 (DS 0.9, IEC 1.61 mmol g-1) and SC-SPAE100 (DS 1.0, IEC 1.74 mmol g-1) reach 0.12 and 0.14 S/cm, respectively, comparable to that of Nafion117 (0.14 S/cm). [ABSTRACT FROM AUTHOR]

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1. [ABSTRACT FROM AUTHOR]

The glutamic acid residue of the conserved PEWY motif of the Qo site of cytochrome bC1 is widely discussed as central to reversible Qo site catalysis of two-electron, two-proton hydroquinone-quinone oxidation-reduction. Extensive mutation of this glutamate (E295) to A, V, F, H, K, and Q in purple photosynthetic Rhodobacter capsulatus results in hydroquinone oxidation rates that are between 5 and 50-fold slower than that in the wild type. However, the mutants show little or no detectable effects on hydroquinone or quinone exchange and binding at the Qo site nor on subsequent Qo site-mediated redox equilibria in the c-chain and b-chain from pH 5–10. Lack of effects of mutations on the Em/pH plots rules out involvement of E295 in the strong electron-proton coupling evident in either the FeS center or heme bL. These detailed equilibrium and kinetic analyses demonstrate that E295 is not irreplaceable in the Qo site catalytic mechanism. Rather, E295 and several other Qo site residues that can also be widely varied and still support hydroquinone oxidation illustrate the considerable resilience of Qo site activity to mutational change in Qo site environs. Residues and water molecules appear to cooperate in providing a physical and chemical environment supporting hydroquinone oxidation rates comparable to those seen in nonprotein aqueous environments at electrodes. We suggest that residues at the Qo site (and, possibly, other respiratory and photosynthetic quinone and oxygen binding sites) are a product of natural selection primarily acting not to lower catalytic barriers according to the traditional view of enzymatic catalysis but rather to develop specificity by raising barriers in defense of semiquinone loss or energy wasting short-circuit reactions. [ABSTRACT FROM AUTHOR]

The photophysics and photochemistryof thioacetamide (CH3CSNH2) after excitationto the S2electronicstate were investigated by using resonance Raman spectroscopy in conjunctionwith the complete active space self-consistent field (CASSCF) methodand density functional theory (DFT) calculations. The A-band resonanceRaman spectra in acetonitrile, methanol, and water were obtained at299.1, 282.4, 266.0, 252.7, and 245.9 nm excitation wavelengths toprobe the structural dynamics of thioacetamide in the S2state. CASSCF calculations were done to determine the transitionenergies and structures of the lower-lying excited states, the conicalintersection points CI(S2/S1) and CI(S1/S0), and intersystem crossing points. The structuraldynamics of thioacetamide in the S2state was revealedto be along eight Franck–Condon active vibrational modes ν15, ν11, ν14, ν10, ν8, ν12, ν18, and ν19, mostly in the CC/CS/CN stretchesand the CNH8,9/CCH5,6,7/CCN/CCS in-planebends as indicated by the corresponding normal mode descriptions.The S2→ S1decay process via the S2/S1conical intersection point as the major channelwere excluded. The thione–thiol photoisomerization reactionmechanism of thioacetamide via the S2,FC→ S′1,minexcited state proton transfer (ESPT) reaction channelwas proposed. [ABSTRACT FROM AUTHOR]

The biosynthetic pathway of diisonitrile chalkophore SF2768 was identified in Streptomyces thioluteus through heterologous expression recently. Isolation and structure elucidation of the N-substituted formamides that coexisted with the diisonitriles implied that a hydration event was involved. In vitro enzymatic assays of an endogenous isonitrile hydratase suggested a rare sequential-hydration of the diisonitriles. Additionally, the results of Cu-CAS assays indicate that both partial and complete hydration led to the loss of the copper-chelating ability of SF2768. [ABSTRACT FROM AUTHOR]

Pyrazolofluostatins A-C (1-3), three new benzo[a]fluorenes with an unprecedented carbon skeleton, were obtained from the South China Sea-derived Micromonospora rosaria SCSIO N160. Their structures were elucidated by extensive spectroscopic analyses. The structure of pyrazolofluostatin A (1) was confirmed by X-ray crystallographic analysis. Notably, 1-3 possessed a benzo[cd]indeno[2,1-f]indazol skeleton with a pyrazole-fused 6/5/6/6/5 pentacyclic ring system. Pyrazolofluostatin A (1) showed moderate antioxidation activity (EC50 48.6 µM). [ABSTRACT FROM AUTHOR]

The immunosuppressive agent caerulomycin A features a unique 2,2′-bipyridine core structure and an unusual oxime functionality. Genetic and biochemical evidence confirms that the oxime formation in caerulomycin A biosynthesis is catalyzed by CrmH, a flavin-dependent two-component monooxygenase that is compatible with multiple flavin reductases, from a primary amine via a N-hydroxylamine intermediate. Structure homologue-guided site-directed mutagenesis studies identify four amino acid residues that are essential for CrmH catalysis. This study provides the first biochemical evidence of a two-component monooxygenase that catalyzes oxime formation. [ABSTRACT FROM AUTHOR]

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. [ABSTRACT FROM AUTHOR]

Xiamycin A (XMA) and oxiamycin (OXM) are bacterial indolosesquiterpenes featuring rare pentacyclic ring systems and are isolated from a marine-derived Streptomyces sp. SCSIO 02999. The putative biosynthetic gene cluster for XMA/OXM was identified by a partial genome sequencing approach. Eighteen genes were proposed to be involved in XMA/OXM biosynthesis, including five genes for terpene synthesis via a non-mevalonate pathway, eight genes encoding oxidoreductases, and five genes for regulation and resistance. Targeted disruptions of 13 genes within the xia gene cluster were carried out to probe their encoded functions in XMA/OXM biosynthesis. The disruption of xiaK, encoding an aromatic ring hydroxylase, led to a mutant producing indosespene and a minor amount of XMA. Feeding of indosespene to XMA/OXM nonproducing mutants revealed indosespene as a common precursor for XMA/OXM biosynthesis. Most notably, the flavin dependent oxygenase XiaI was biochemically characterized in vitro to convert indosespene to XMA, revealing an unusual oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis. [ABSTRACT FROM AUTHOR]

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangiuni aurantiacuin subsp. hamdenensis NRRL 18085. Sequence analysis of a 110633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaGi and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P4SOs (TiaPi and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis. [ABSTRACT FROM AUTHOR]

A novel pseudorotaxane of cucurbit[6]uril (CB[6]) and N,N′-hexamethylenebis (pyrazinyl bromide) was synthesized and characterized by 1H NMR, thermogravimetric analysis, and single crystal X-ray diffraction analysis. The crystal structure of pseudorotaxane was described, which self-assembled via hydrogen bonds of (H 2O) 3clusters and formed two types of linear structures. Furthermore, Br −(H 2O) 3anion clusters can serve as bridges to associate these linear structures and form three-dimensional networked pseudopolyrotaxane. [ABSTRACT FROM AUTHOR]