Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.     

Synthesis of de novo designed small-molecule inhibitors of bacterial RNA polymerase

The de novo molecular design program SPROUT has been used in conjunction with the X-ray crystal structure of RNA polymerase (RNAP) from Thermus aquaticus to produce a novel enzyme inhibitor scaffold. A short and efficient synthesis of molecules corresponding to this scaffold has been developed and in keeping with the design predictions, the resulting inhibitors displayed useful levels of inhibition of Escherichia coli RNAP.     

Labeling substrates of protein arginine methyltransferase with engineered enzymes and matched S-adenosyl-L-methionine analogues

Elucidating physiological and pathogenic functions of protein methyltransferases (PMTs) relies on knowing their substrate profiles. S-adenosyl-L-methionine (SAM) is the sole methyl-donor cofactor of PMTs. Recently, SAM analogues have emerged as novel small-molecule tools to efficiently label PMT substrates. Here we reported the development of a clickable SAM analogue cofactor, 4-propargyloxy-but-2-enyl SAM, and its implementation to label substrates of human protein arginine methyltransferase 1 (PRMT1). In the system, the SAM analogue cofactor, coupled with matched PRMT1 mutants rather than native PRMT1, was shown to label PRMT1 substrates. The transferable 4-propargyloxy-but-2-enyl moiety of the SAM analogue further allowed corresponding modified substrates to be characterized through a subsequent click chemical ligation with an azido-based probe. The SAM analogue, in combination with a rational protein-engineering approach, thus shows potential to label and identify PMT targets in the context of a complex cellular mixture.     

The use of acrylic acid-co-nylon substrates for immobilizing enzymes

A number of poly(acrylic acid)-co-nylon graft copolymers were prepared. Two approaches were used. The first involved the direct, radiation-induced grafting of acrylic acid onto the nylon, while the second involved the hydrolysis of polyacrylonitrile-co-nylon graft copolymers. All the resulting poly(acrylic acid)-co-nylon graft copolymers coupled large amounts of protein when the carbodimides CMC and EDAC were used as coupling agents. However, when the enzymes β-galactosidase and trypsin were immobilized, the retention of activity was relatively low. A number of reasons for this effect can be postulated, some of which were investigated. From the evidence, it would appear that the major reason is that the carboxylic acid groups are fixed to a “rigid” backbone, which allows them to interact with the enzyme after the establishment of an initial covalent attachment. Additionally, with acid phosphatase, it was observed that the enzyme interacted with immobilized carboxylic acid groups before coupling occurred.     

A deoxyribozyme that forms a three-helix-junction complex with its RNA substrates and has general RNA branch-forming activity

We recently used in vitro selection to identify 7S11, a deoxyribozyme that synthesizes 2',5'-branched RNA. The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. Here, we describe 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry. First, on the basis of a comprehensive analysis of its substrate sequence requirements, 7S11 is shown to be generally applicable for the synthesis of a wide range of 2',5'-branched RNAs. Strict substrate sequence requirements are found at the two RNA nucleotides that directly form the branched linkage, and these requirements correspond to those nucleotides found most commonly at these two positions in natural spliced RNAs. Outside of these two nucleotides, most substrate sequences are tolerated with useful ligation activity, although rates and yields vary. Because chemical synthesis approaches to branched RNA are extremely limited in scope, the deoxyribozyme-based route using 7S11 will enable many experiments that require branched RNA. Second, comprehensive nucleotide covariation experiments demonstrate that 7S11 and its RNA substrates adopt a three-helix-junction structure in which the branch-site nucleotide is located at the intersection of the three helices. Because many natural ribozymes have multi-helix junctions, 7S11 is an interesting model system for catalytic nucleic acids.     

An aptazyme-based molecular device that converts a small-molecule input into an RNA output

We describe the construction of an aptazyme-based molecular device that converts, through a cascade of reactions, a small-molecule input into output RNA strands. This device is applicable as an interface between a small molecule and a molecular system that accepts only nucleic acid input.     

Scanning electrochemical microscopy of enzymes immobilized on structured glass-gold substrates

Scanning electrochemical microscopy (SECM) was used to characterize enzyme-modified glass-gold specimens. The exposed gold surface was functionalized with an aminothiol and reacted with carbodiimide-activated glucose oxidase. The specimen surface was examined with SECM, using a 25 μm platinum electrode. Images were acquired showing the topography, electric conductivity, and enzymatic activity of the composite surface. It was found that the hydroxy-groups of the glass surface are as likely to bind to the activated enzyme as the amino-groups on the gold surface.     

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

Background: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5′-5′-pyrophosphate ‘capped’ RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5′-monophosphate (AMP) may be a vestige of ‘RNA world’ catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.Results: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10¹⁵ RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5′-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of ∼ 5 × 10⁵ over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3′-5′-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.Conclusions: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.     

Treatment of Alzheimer's disease with small-molecule photosensitizers

Alzheimer’s disease is a neurodegenerative disease that signals for excess β-amyloid(Aβ) aggregation.Although people have made great attempts to control the aggregation of Aβ, no effective medications have been produced yet. Due to its excellent temporal and spatial selectivity, photodynamic treatment has been gradually employed and interfered in the aggregation process of Aβ, with some achievement. To enhance the research and application of photodynamic therapy in Alzheimer’s disease, this pape...     

Catalytic oxidations of steroid substrates by artificial cytochrome p-450 enzymes

Catalysts comprising manganese-porphyrins carrying cyclodextrin binding groups are able to perform hydroxylations with substrate selectivity and regio- and stereoselectivity and high catalytic turnovers. The geometries of the catalyst/substrate complexes override intrinsic substrate reactivities, permitting attack on geometrically accessible saturated carbons of steroids in the presence of secondary carbinol groups and carbon-carbon double bonds, as in enzymatic reactions. Selective hydroxylations of steroid carbon 9 positions are of particular practical interest.     

Coalescence of two drops on partially wettable substrates

The present work contains the results of the experiments with two tiny drops on partially wettable substrates with contact angles of 10°, 24°, 27°, and 56°, which coalesce in the regime entirely dominated by viscous forces. Both side and bottom views are examined. The results for these three-dimensional coalescence flows are compared with scaling laws and the numerical two-dimensional model developed in the present work.     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

Solid-state fermentation of new substrates for production of cellulase and other biopolymer-hydrolyzing enzymes

Applied Biochemistry and Biotechnology -     

UV protective effects of DNA repair enzymes and RNA lotion

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes ( Micrococcus luteus ) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.     

Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: α-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided withde novo thiol-groups by a two-step reaction involvingN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases. In order to interrupt rapidly the reactions in which the enzymes were used, the modified enzyme was immobilized by reaction via its thiol groups on a thiolsulfinate-agarose derivative. The gel-bound enzyme could then be easily removed from unreacted substrate and product by filtration or centrifugation. Comparative studies showed that the immobilized enzymes had much lower activities in the reactions studied than the corresponding soluble ones. The potential for enzyme reuse was also demonstrated with the a-amylase derivatives, which were quantitatively released and eluted in fully active form from the agarose. We have shown that it is possible to achieve practically complete enzyme immobilization in short times and thus to control the progress of the reactions. Because of its simplicity and high efficiency, this approach may represent an interesting alternative for biotechnological processes involving macromolecular or solid substrates.     

Visualizing deeper into the body with a NIR-Ⅱ small-molecule fluorophore

正Fluorescence imaging in the near-infrared(NIR)window(750–900 nm)is one of the most popular work modes in biomedical sciences and clinical procedures for visualization of cells and tissues.Up to date,only indocyanine green(ICG)and methylene blue have been approved by the FDA