Artificial Splitting of a Non‐Ribosomal Peptide Synthetase by Inserting Natural Docking Domains

The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme.     

Sequential In Vitro Cyclization by Cytochrome P450 Enzymes of Glycopeptide Antibiotic Precursors Bearing the X‐Domain from Nonribosomal Peptide Biosynthesis

The biosynthesis of the glycopeptide antibiotics, which include vancomycin and teicoplanin, relies on the interplay between the peptide‐producing non‐ribosomal peptide synthetase (NRPS) and Cytochrome P450 enzymes (P450s) that catalyze side‐chain crosslinking of the peptide. We demonstrate that sequential in vitro P450‐catalyzed cyclization of peptide substrates is enabled by the use of an NRPS peptide carrier protein (PCP)‐X di‐domain as a P450 recruitment platform. This study reveals that whilst the precursor peptide sequence influences the installation of the second crosslink by the P450 OxyA_tei, activity is not restricted to the native teicoplanin peptide. Initial peptide cyclization is possible with teicoplanin and vancomycin OxyB homologues, and the latter displays excellent activity with all substrate combinations tested. By using non‐natural X‐domain substrates, bicyclization of hexapeptides was also shown, which demonstrates the utility of this method for the cyclization of varied peptide substrates in vitro.     

Structural Basis for the Identification of an i‐Motif Tetraplex Core with a Parallel‐Duplex Junction as a Structural Motif in CCG Triplet Repeats

CCG triplet repeats can fold into tetraplex structures, which are associated with the expansion of (CCG)_n trinucleotide sequences in certain neurological diseases. These structures are stabilized by intertwining i‐motifs. However, the structural basis for tetraplex i‐motif formation in CCG triplet repeats remains largely unknown. We report the first crystal structure of a CCG‐repeat sequence, which shows that two dT(CCG)₃A strands can associate to form a tetraplex structure with an i‐motif core containing four C:C⁺ pairs flanked by two G:G homopurine base pairs as a structural motif. The tetraplex core is attached to a short parallel‐stranded duplex. Each hairpin itself contains a central CCG loop in which the nucleotides are flipped out and stabilized by stacking interactions. The helical twists between adjacent cytosine residues of this structure in the i‐motif core have an average value of 30°, which is greater than those previously reported for i‐motif structures.     

Discovery of a Single Monooxygenase that Catalyzes Carbamate Formation and Ring Contraction in the Biosynthesis of the Legonmycins

Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD‐dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so‐called legonmycins. By genome‐driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non‐ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time.     

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin‐Sensing Riboswitch

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin‐sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch–ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.     

Structural Basis for Bulky‐Adduct DNA‐Lesion Recognition by the Nucleotide Excision Repair Protein Rad14

Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8‐guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion‐detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures.     

Non‐Covalent Functionalization of Graphene with Bisphenol A for High‐Performance Supercapacitors

The reduced graphene oxide (RGO)/bisphenol A (BPA) composites were prepared by an adsorption‐reduction method. The composites are characterized by X‐ray diffraction (XRD), UV‐vis, thermogravimetric (TG) analysis, field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM). The results confirm that BPA is adsorbed on the basal plane of RGO by π‐π stacking interaction. Furthermore, the electrochemical behaviors were evaluated by cyclic voltammetry, galvanostatic charge/discharge techniques and electrochemical impedance spectroscopy (EIS). The results show that the RGO/BPA nanocomposites exhibit ultrahigh specific capacitance of 466 F·g^?1 at a current density of 1 A·g^?1, excellent rate capability (more than 81% retention at 10 A·g^?1 relative to 1 A·g^?1) and superior cycling stability (90% capacitance decay after 4000 cycles). Consequently, the RGO/BPA nanocomposites can be regarded as promising electrode materials for supercapacitor applications.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

A density functional theory study on the catalytic mechanism of hydroxycinnamoyl‐CoA hydratase‐lyase

Hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL), a particular member of the crotonase superfamily, catalyzes the bioconversion of feruloyl‐CoA to the important flavor and fragrance compound vanillin. In this article, the catalytic mechanism of HCHL has been studied by using hybrid density functional theory method with simplified models. The calculated results reveal that the mechanism involves the hydration of the C?C double bond of feruloyl‐CoA and thence the cleavage of C? C single bond of β‐hydroxythioester. The hydration step is a typical concerted process, whereas C? C bond cleavage follows a concerted but asynchronous mechanism. The calculated energy barrier of hydration reaction is only slightly lower than that of cleavage process, implying both of two processes are rate limiting. By using three substrate analogs, the substrate specificity of HCHL was further examined. It is found that the p‐hydroxyl group of aromatic ring is necessary for the catalytic reaction. © 2013 Wiley Periodicals, Inc.     

Isolation and Structural Elucidation of Novel Mycosporine‐Like Amino Acids as Alarm Cues in the Defensive Ink Secretion of the Sea Hare Aplysia californica

Three new mycosporine‐like amino acids (MAAs), aplysiapalythines A, B, and C ( 1, 2 , and 3 , resp.) were isolated from opaline, a glandular component of the defensive ink secretion of sea hares (Aplysia californica) collected from waters off southern California. Here, we report the structure of these MAAs determined by mass spectrometry and NMR data. These new MAAs are structurally related to two known MAAs that are also present in sea hare opaline, i.e., asterina 330 ( 4 ) and palythine ( 5 ), and for which we also provide detailed data here for comparison. The fact that three of the five MAAs that we identified from sea hare opaline are novel molecules is interesting given that this represents a relatively large addition to the current list of known MAAs. This is likely because most researchers have identified MAAs through HPLC with UV detection, which is imprecise given similarities in UV spectra for different MAAs. Our findings suggest that there is much greater diversity of MAAs than is currently known. Results published elsewhere show that 1, 2 , and 4 are alarm cues for conspecific sea hares at natural concentrations, but 3 and 5 are not.     

Experimental and DFT Evidence for the Fractional Non‐Innocence of a β‐Diketonate Ligand

New compounds [Ru(pap)₂(L)](ClO₄), [Ru(pap)(L)₂], and [Ru(acac)₂(L)] (pap=2‐phenylazopyridine, L^?=9‐oxidophenalenone, acac^?=2,4‐pentanedionate) have been prepared and studied regarding their electron‐transfer behavior, both experimentally and by using DFT calculations. [Ru(pap)₂(L)](ClO₄) and [Ru(acac)₂(L)] were characterized by crystal‐structure analysis. Spectroelectrochemistry (EPR, UV/Vis/NIR), in conjunction with cyclic voltammetry, showed a wide range of about 2 V for the potential of the Ru^III/II couple, which was in agreement with the very different characteristics of the strongly π‐accepting pap ligand and the σ‐donating acac^? ligand. At the rather high potential of +1.35 V versus SCE, the oxidation of L^? into L^. could be deduced from the near‐IR absorption of [Ru^III(pap)(L^.)(L^?)]²⁺. Other intense long‐wavelength transitions, including LMCT (L^?→Ru^III) and LL/CT (pap^.?→L^?) processes, were confirmed by TD‐DFT results. DFT calculations and EPR data for the paramagnetic intermediates allowed us to assess the spin densities, which revealed two cases with considerable contributions from L‐radical‐involving forms, that is, [Ru^III(pap⁰)₂(L^?)]²⁺?[Ru^II(pap⁰)₂(L^.)]²⁺ and [Ru^III(pap⁰)(L^?)₂]⁺?[Ru^II(pap⁰)(L^?)(L^?)]⁺. Calculations of electrogenerated complex [Ru^II(pap^.?)(pap⁰)(L^?)] displayed considerable negative spin density (?0.188) at the bridging metal.     

Structural Characterization and Enzymatic Degradation of α‐, β‐, and γ‐Crystalline Forms for Poly(β‐propiolactone)

A structural comparison of three different crystalline forms of poly(β‐propiolactone) (PPL) was carried out by wide‐angle X‐ray diffraction, Fourier‐transform infrared spectroscopy, and differential scanning calorimetry. The α‐form in a hot‐drawn and annealed film represents a 2₁ helix conformation. The β‐form in a cold‐drawn and annealed film represents a planar zigzag conformation. The γ‐form in an oriented sedimented mat of solution‐grown chain‐folded lamellar crystals also implies a planar zigzag conformation. The solution‐cast film depicts similar outlines with the γ‐form in lamellar crystals in all the experimental measurements, suggesting that the molecular chain in the solution‐cast film has a planar zigzag conformation. While elongation at break decreased, tensile strength and Young's modulus increased with an increase in the crystallinity, independent of the crystalline forms. The influence of the enzymatic degradation of these crystal structures has been investigated by using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The rate of degradation was in the order of β‐form > α‐form > solution‐cast (γ‐form) film, and the different surface morphologies after partial enzymatic degradation were observed in scanning electron micrographs. It is suggested that the crystal structure is one of the important factors for determining the rate of degradation together with crystallinity.

Enzymatic degradation profiles of poly(β‐propiolactone) films.     

Synergistic Interplay of a Non‐Heme Iron Catalyst and Amino Acid Coligands in H2O2 Activation for Asymmetric Epoxidation of α‐Alkyl‐Substituted Styrenes

Highly enantioselective epoxidation of α‐substituted styrenes with aqueous H₂O₂ is described by using a chiral iron complex as the catalyst and N‐protected amino acids (AAs) as coligands. The amino acids synergistically cooperate with the iron center in promoting an efficient activation of H₂O₂ to catalyze epoxidation of this challenging class of substrates with good yields and stereoselectivities (up to 97 % ee) in short reaction times.     

A New Strategy for Fluorogenic Esterase Probes Displaying Low Levels of Non‐specific Hydrolysis

A new design for fluorescence probes of esterase activity that features a carboxylate‐side pro‐fluorophore is demonstrated with boron dipyrromethene (BODIPY)‐based probes 1 a and 1 b . Because the design relies on the enzyme‐catalyzed hydrolysis of an ester group that is not electronically activated, these probes exhibit a stability to background hydrolysis that is far superior to classical alcohol‐side profluorophore‐based probes, large signal‐to‐noise ratios, reduced sensitivity to pH variations, and high enzymatic reactivity. The utility of probe 1 a was established with a real‐time fluorescence imaging experiment of endogenous esterase activity that does not require washing of the extracellular medium.     

N,N,N‐Trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate a precursor for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[18F]‐fluoronicotinate

The synthesis, recrystallization, and X‐ray deterimination of N,N,N‐trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate (PyTFP‐precursor), C₁₅H₁₃F₄N₂O₂⁺·CF₃SO₃⁻, is described. This triflate salt precursor is required for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[¹⁸F]‐fluoronicotinate ([¹⁸F]FPyTFP), a prosthetic group used to radiolabel peptides for positron emission tomography (PET), as peptides are increasingly being used as PET‐imaging probes in nuclear medicine. Radiolabeling of peptides is typically done using a `prosthetic group', a small synthon to which the radioisotope is attached in the first step, followed by attachment to the peptide in the second step. During the synthesis of the PyTFP‐precursor, displacement of a Cl atom with trimethylamine gas and anion replacement with a triflate counter‐ion is critical, as incomplete replacement would hinder radioisotopic incorporation of nucleophilic fluorine‐18 and result in diminished radiochemical yields. The structural determination of the PyTFP‐precursor by X‐ray crystallography helped confirm the anion exchange of chloride with triflate.