Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual "glycolate" chain extension unit in ansamitocin biosynthesis

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-13C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster.     

A facile and efficient method for the incorporation of multiple unnatural amino acids into a single protein

One stone, two birds: Here, we have developed a simple and efficient method for the incorporation of multiple unnatural amino acids in a single protein. This single protein exhibited two different novel functionalities acquired from the genetically incorporated unnatural amino acids, which is an interesting and not an inherent property of the protein.     

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.     

Kinetics of copper incorporation into a biosynthetic purple Cu(A) azurin: characterization of red, blue, and a new intermediate species

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.     

Soluble aromatic polyamides modified by incorporation of 1,2,4-triazole and pentadecyl units into the backbone of polymer

Abstract

A new series of soluble aromatic polyamides was synthesized by low temperature solution polycondensation of novel aromatic diamine namely 3,5-bis-(4′-amino phenyl)-4-(4″-methoxy-2″-pentadecyl phenyl) 1,2,4-triazole (VII) with aromatic diacid chlorides, viz. isophthaloyl chloride (IPC) and terephthaloyl chloride (TPC). The aromaticdiamine (VII) was characterized by elemental analysis, FT-IR, NMR (¹H, ¹³C), and mass spectrometry. Copolyamides were also synthesized by employing various mole proportions of IPC and TPC with diamine (VII). Inherent viscosities of these polyamides were in the range of 0.50–0.65 dL/g in DMAc, indicating formation of moderate to high molecular weight of polyamides. These polyamides showed good solubility in polar aprotic solvents such as N,N-Dimethyl acetamide (DMAc), N-Methyl 2-pyrrolidone (NMP), N, N, Dimethyl formamide (DMF), and Dimethyl sulphoxide (DMSO), which may be due to incorporation of pendant methoxyphenyl moiety with pentadecyl units. The amorphous morphology of polyamides as evidenced by XRD. These polyamides had lower glass transition temperatures; as determined by DSC, compared to the T_g of conventional aromatic polyamides due to internal plasticization effect of long alkyl pentadecyl group. Polymers showed good thermal stability, with initial decomposition temperature above 300?°C.     

Premalbrancheamide: synthesis, isotopic labeling, biosynthetic incorporation, and detection in cultures of Malbranchea aurantiaca

An advanced metabolite, named premalbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double (13)C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, premalbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed.     

Structure,biosynthetic origin,and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.     

Studies on the biosynthesis of the notoamides: synthesis of an isotopomer of 6-hydroxydeoxybrevianamide E and biosynthetic incorporation into notoamide J

6-Hydroxydeoxybrevianamide E is proposed as a biosynthetic precursor to several advanced metabolites isolated from both marine-derived Aspergillus sp. and a terrestrial-derived Aspergillus versicolor. To verify the role of this reverse-prenylated indole alkaloid as an intermediate along the biosynthetic pathway, [(13)C](2)-[(15)N]-6-hydroxydeoxybrevianamide E was synthesized and fed to Aspergillus versicolor. Analysis of the metabolites showed incorporation of the intermediate only into the natural product notoamide J.     

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidin Earlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3) . To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12-[³H]-farnesol as an internal standard: 2-[¹⁴C]-, 7-[¹⁴C]-, 7′, 8-[¹⁴C]-, 7′, 8-[³H]-geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U-[¹⁴C]-leucine, U-[¹⁴C]-isoleucine and 1-[¹⁴C]-glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1-[¹⁴C]- and 2-[¹⁴C]-glycine do not serve as precursors for cantharidin (1) .     

Insight into the molecular mechanism of yeast acetyl-coenzyme A carboxylase mutants F510I,N485G,I69E,E477R,and K73R resistant to soraphen A

Acetyl-coenzyme A carboxylases (ACCs) is the first committed enzyme of fatty acid synthesis pathway. The inhibition of ACC is thought to be beneficial not only for diseases related to metabolism, such as type-2 diabetes, but also for infectious disease like bacterial infection disease. Soraphen A, a potent allosteric inhibitor of BC domain of yeast ACC, exhibit lower binding affinities to several yeast ACC mutants and the corresponding drug resistance mechanisms are still unknown. We report here a theoretical study of binding of soraphen A to wild type and yeast ACC mutants (including F510I, N485G, I69E, E477R, and K73R) via molecular dynamic simulation and molecular mechanics/generalized Born surface area free energy calculations methods. The calculated binding free energies of soraphen A to yeast ACC mutants are weaker than to wild type, which is highly consistent with the experimental results. The mutant F510I weakens the binding affinity of soraphen A to yeast ACC mainly by decreasing the van der Waals contributions, while the weaker binding affinities of Soraphen A to other yeast ACC mutants including N485G, I69E, E477R, and K73R are largely attributed to the decreased net electrostatic (ΔE_ele?+?ΔG_GB) interactions. Our simulation results could provide important insights for the development of more potent ACC inhibitors.     

A new class of iridium complexes suitable for stepwise incorporation into linear assemblies: synthesis, electrochemistry, and luminescence

A new family of cationic iridium(III) complexes is reported that contain two cyclometalating terdentate ligands. The complex [Ir(N--C--N-dpyx)(N--N--C-phbpy)]+ (1) contains one N--C--N-coordinating ligand, cyclometalating through the central phenyl ring, and one N--N--C-coordinated ligand, cyclometalated at the peripheral phenyl ring [dpyxH = 1,3-di(2-pyridyl)-4,6-dimethylbenzene; phbpyH = 6-phenyl-2,2'-bipyridine]. This binding mode dictates a mutually cis arrangement of the cyclometalated carbon atoms: the complexes are thus bis-terdentate analogues of the well-known [Ir(N--C-ppy)2(N--N-bpy)]+ family of complexes, which similarly contain a cis-C2N4 coordination environment. The dpyx ligand can be brominated regioselectively at the carbon atom para to the metal under mild conditions. Starting from a modified complex, [Ir(N--C--N-dpyx)(N--N--C-mtbpy-phi-Br)]+ (2), which incorporates a pendent bromophenyl group, a sequential cross-coupling-bromination-cross-coupling strategy can be applied for the stepwise introduction of aryl groups into the ligands, using in situ palladium-catalyzed Suzuki reactions with arylboronic acids [mtbpyH-phi-Br = 4-(p-bromophenyl)-6-(m-tolyl)bipyridine]. Dimetallic complexes 6 and 7 have similarly been prepared by a palladium-catalyzed reaction of complex 2 with 1,4-benzenediboronic acid and 4,4'-biphenyldiboronic acid, respectively. All five monometallic complexes and both dimetallic systems are luminescent in solution, emitting around 630 nm in MeCN at 298 K, with quantum yields in the range of 0.02-0.06, superior to [Ir(ppy)2(bpy)]+. The luminescence, electrochemistry, and singlet-oxygen-sensitizing abilities of the new family of complexes are discussed in the context of the tris-bidentate analogues and related bis-terdentate compounds that contain a trans arrangement of cyclometalated carbon atoms.     

On the incorporation of the non-steroidal anti-inflammatory naproxen into cationic O/W microemulsions

Microemulsions (ME) containing hexadecyltrimethylammonium bromide (HTAB)/ethanol as surfactant, isopropylmyristate (IM) or butylstearate (BS) as oil phase and aqueous buffer were studied. Pseudo-ternary phase diagrams of the investigated systems were obtained at constant surfactant/cosurfactant molar ratio (1:5) by titration in order to characterize the proportions between the components to obtain clear systems. Oil in water microemulsions were prepared in a wide range of phase volume (phi). UV-vis absorption spectra of naproxen at pH 5.5 showed that the solubility of Np increases significantly in the presence of O/W ME in high phase volumes. For both, IM and BS microemulsions, the dynamic light scattering experiments showed that the size of the oil droplets remains constant in low values of phi, increasing abruptly in high phi values. Phase solubility study revealed that for both IM and BS microemulsions, the drug incorporation followed a straight-line profile in all range of phi. The data could be analyzed through the phase-separation model and the association constants (K) calculated varied from 27 to 90 M(-1), depending on the pH and on the microemulsion oil phase.     

A common mechanism for branching, cyclopropanation, and cyclobutanation reactions in the isoprenoid biosynthetic pathway

Four reactions--chain elongation, cyclopropanation, branching, and cyclobutanation--are used in nature to join isoprenoid units for construction of the carbon skeletons for over 55,000 naturally occurring isoprenoid compounds. Those molecules produced by chain elongation have head-to-tail (regular) carbon skeletons, while those from cyclopropanation, branching, or cyclobutanation have non-head-to-tail (irregular) skeletons. Although wild type enzymes have not been identified for the branching and cyclobutanation reactions, chimeric proteins constructed from farnesyl diphosphate synthase (chain elongation) and chrysanthemyl diphosphate synthase (cyclopropanation) catalyze all four of the known isoprenoid coupling reactions to give a mixture of geranyl diphosphate (chain elongation), chrysanthemyl diphosphate (cyclopropanation), lavandulyl diphosphate (branching), and maconelliyl and planococcyl diphosphate (cyclobutanation). Replacement of the hydrogen atoms at C1 or C2 or hydrogen atoms in the methyl groups of dimethylallyl diphosphate by deuterium alters the distribution of the cyclopropanation, branching, and cyclobutanation products through primary and secondary kinetic isotope effects on the partitioning steps of common carbocationic intermediates. These experiments establish the sequence in which the intermediates are formed and indicate that enzyme-mediated control of the carbocationic rearrangement and elimination steps determines the distribution of products.     

A new incorporation mechanism for trivalent actinides into bioapatite: a TRLFS and EXAFS study

One of the most toxic byproducts of nuclear power and weapons production is the transuranics, which have a high radiotoxicity and long biological half-life due to their tendency to accumulate in the skeletal system. This accumulation is inhomogeneous and has been associated with the chemical properties and structure of the bone material rather than its location or function. This suggests a chemical driving force to incorporation and requires an atomic scale mechanistic understanding of the incorporation process. Here we propose a new incorporation mechanism for trivalent actinides and lanthanides into synthetic and biologically produced hydroxyapatite. Time-resolved laser fluorescence spectroscopy and extended X-ray absorption fine structure have been used to demonstrate that trivalent actinides and lanthanides incorporate into the amorphous grain boundaries of apatite. This incorporation site can be used to explain patterns in uptake and distribution of radionuclides in the mammalian skeletal system.